Session 3. Proteins — Structure and Function Lectures L3.1 L3.2

Transkrypt

Session 3. Proteins — Structure and Function
Lectures
L3.2
L3.1
The mechanism of RNase H activity in
DNA repair and reverse transcription
Crystal structures of two neurotoxic
phospholipases A2 and identification
of their interaction sites with
various biological targets
Marcin Nowotny
Grazyna Faure
International Institute of Molecular and Cell Biology, Laboratory of
Protein Structure, Warsaw, Poland
Institut Pasteur, Unité Récepteurs – Canaux, Department of
Neuroscience, France
e-mail: Marcin Nowotny <[email protected]>
e-mail: Grazyna Faure <[email protected]>
RNases H are nucleases that cleave ribonucleotides in
RNA/DNA hybrid duplexes. Two classes of these enzymes have been described — RNases H1 and RNases H2.
The first requires a stretch of at least four ribonucleotides
in the hybrid for the cleavage to occur. In addition to its
cellular form it also exists as a domain of reverse transcriptases — enzymes converting single-stranded RNA to
double-stranded DNA. Reverse transcriptases participate
proliferation of retrotransposons and retroviruses such
as HIV and RNase H activity is essential for this process.
The second type is RNase H2 which specifically cleaves
single ribonucleotides embedded in the DNA. Such single
ribonucleotides are mistakenly incorporated by DNA polymerases and lead to genome instability. Therefore, ribonucleotide excision by RNase H2 protects the genomic DNA.
In the talk the mechanisms of various RNases H will be
discussed as elucidated based on crystal structures of
RNases H2 and reverse transcriptases complexed with their
nucleic acid substrates. RNase H2 utilizes a unique mechanism in which two divalent metal ions are coordinated at
the active site. The RNA-DNA junction present in the substrate is specifically deformed so that its phosphate group
participates in metal ion coordination. Therefore, only in
the presence of the proper substrate the metal ions can
be bound and promote catalysis which assures enzyme’s
specificity.
For reverse transcriptases an important part of their mechanism is the regulation of the RNase H activity. Our crystallographic and SAXS studies showed that for example in
the monomeric reverse transcriptase from XMRV virus the
RNase H domain is very mobile and gets organized only
when the proper RNA/DNA substrate is present. Very different mechanisms exist for dimeric reverse transcriptases
such as the HIV enzyme and the enzymes from retrotransposons. These mechanisms will also be discussed.
Snake venoms are a rich source of phospholipases A2
(PLA2) with distinct biological functions and with a high
potential for medical and pharmaceutical applications. We focus here on structural and functional studies of two
neurotoxic PLA2, the monomeric ammodytoxin from Vipera ammodytes ammodytes, and heterodimeric crotoxin from
Crotalus durissus terrificus. Recently we have determinedat
high resolutiontheirthree-dimensional structures. These
two enzymes are i) potent β-neurotoxins which target neuronal or peripheral presynapic receptors and ii) potent anticoagulant molecules, which directly interact with human
coagulation factor Xa. Comparative structural analysis of
PLA2 isoforms allowed us to detect local conformational
changes and delineate the role of critical residues in the
anticoagulant and neurotoxic functions of these neurotoxic
PLA2 (Saul et al., 2010). The crystal structure of crotoxin
revealed details of the binding interface between its acidic
(CA) and basic (CB) subunits and helps us to understand
the role played by critical residues of the CA subunit in
the increased toxicity and stability and reduced enzymatic
activity of the crotoxin complexes (Faure et al., 2011).
More recently, we identified a new target of these PLA2
and described their novel function as a potential pharmacological effector of human Cystic Fibrosis Transmembrane
Regulator (CFTR) Cl- channel activity (Faure et al., submitted). We demonstrated the physical and functional interaction of wild-type and mutated CFTR with PLA2. A 3D
structural model of the complex will be presented. The potentiating and correcting effects of CB on mutated CFTR
(the ΔF508, most frequent mutation responsible of cystic
fibrosis) renders this PLA2 attractive for potential clinical
applications and offers an original perspective to investigate the pharmacotherapy of CF. An overview of these
new data will be presented.
Rreferences:
Saul FA et al (2010) J Struct Biol 169: 360–369.
Faure G et al (2011) J Mol Biol 412: 176–191.
Toruń, September 2nd–5th 2013
34
L3.3
L3.4
Structural and thermodynamic studies of
ligand binding by bovine β-lactoglobulin
Monster crystal structure with 28
independent protein molecules
Krzysztof Lewiński1, Joanna Loch1, Piotr
Bonarek2, Agnieszka Polit2
Mariusz Jaskolski1,2, Joanna Sliwiak1, Zbigniew
Dauter3, Airlie McCoy4, Randy J. Read4
1Faculty of Chemistry, Jagiellonian University, Kraków, Poland; 2Faculty
of Biochemistry, Biophysics and Biotechnology, Jagiellonian University,
Kraków, Poland
1Center for Biocrystallographic Research, Institute of Bioorganic
Chemistry, Polish Academy of Sciences, Poznań, Poland; 2Department
of Crystallography, Faculty of Chemistry, A. Mickiewicz University,
Poznań, Poland; 3Synchrotron Radiation Research Section, National
Cancer Institute, Argonne National Laboratory, Argonne, USA;
4Department of Haematology, University of Cambridge, Cambridge, UK
e-mail: Krzysztof Lewiński <[email protected]>
Bovine β-lactoglobulin (LGB), is a dimeric protein being
one of the major components of bovine whey. Due to its
ability to bind wide range of hydrophobic compounds,
LGB is an object of intensive studies, since its discovery in
1934. The primary binding site is located in the centre of
eight-stranded β-barrel and is surrounded by flexible loops.
The conformational change of EF loop at pH around 7.0
regulates access to the binding site. Existence of other
binding sites located on dimer interface was postulated by
some authors but confirmed only in the structure of LGBvitamin D3 complex. Physiological function of LGB has
not been recognized. It is probably involved in transport
of hydrophobic compounds and regulation of enzymatic
activity of pregastric lipase. Potential utilization of modified LGB in clinical applications as molecular transporters requires better understanding of its binding properties
and role of individual residues in the binding site. For that
purpose, we have undertaken systematic structural and
thermodynamic studies of LGB complexes with different ligands. Among studied ligands were 8- to 18-carbon
saturated fatty acids, 18-carbon unsaturated linoleic and
oleic acid as well as dodecyltrimethylammonium chloride
(DTAC) and sodium dodecyl sulfate (SDS).
The crystal structures were typically determined with resolution 2.2–1.9 Å. In all structures ligand molecule was
bound only in the β-barrel. We have find that position of
ligand polar group strongly depended on length of the aliphatic chain, indicating that competition between polar and
hydrophobic interactions is an important factor determining location of ligand in the binding site. It is interesting,
that in case of 12-carbon ligands (lauric acid, SDS, DTAC)
the chemical character of ligand polar group did not affect
the position of its aliphatic chain, indicating that hydrophobic interactions prevailed even with the repulsion between
positively charged DTAC and lysine residues located at the
binding site entrance. The thermodynamic parameters have
been determined by isothermal titration calorimetry. They
revealed that binding of ligands is entropically driven and
entropic component of ΔG increases with elongation of
aliphatic chain.
The complexes of LGB with SDS, DTAC and lauric acid
were studied for two most abundant LGB isoforms, A
and B, to determine influence of substitutions D64G and
V118A on crystal structure and thermodynamics of ligand
binding.
e-mail: Mariusz Jaskolski <[email protected]>
Stress factors, such as pathogens, induce in plants the expression of so-called Pathogenesis-Related (PR) proteins,
which have been divided into seventeen classes. PR proteins of class 10 (PR-10) are mysterious proteins with no
unique biological function attributed to them despite their
abundance and involvement in other processes, such as developmental regulation or symbiosis. Recent results, mostly
from our group, strongly implicate the PR-10 proteins in
phytohormone binding and regulation. The ligand binding
occurs in a large cavity that is present in the hydrophobic core of the protein. PR-10/hormone complexes are
studied inter alia using fluorescent probes, such as ANS. We
have crystallized an ANS complex using a PR-10 protein
from St John’s wort (Hyp-1) with a controversial implication in the biosynthesis of hypericin. Solution of the apparent P4122 crystal structure was impossible by standard
molecular replacement methods because of evident tetartohedral twinning and an extremely bizarre modulation of
the reflection intensities hkl with l periodicity of 7, which
indicated seven-fold non-crystallographic translation along
c. Ultimately, the structure was solved using Phaser after
data expansion to P1 symmetry, with as many as 56 Hyp-1
molecules in the unit cell. Analysis of their arrangement
revealed the true C2 space group with 28 independent
protein molecules, arranged in 14 dimers around a noncrystallographic 21 screw along c with a pitch of 1/7. The
monstrous crystal structure has been successfully refined to
an R-factor of 21.8%, revealing the presence of three ANS
binding sites in each Hyp-1 molecule, two of which are located within the internal cavity, and one in a surface pocket.
There are also numerous interstitial ANS molecules. From
the crystallographic point of view, this is a rare example of
a commensurately modulated protein structure, with sevenfold repetition of the basic unit cell in the c direction. This
crystal structure holds a record in the PDB from the point
of view of the complexity of the asymmetric unit contents.
48th Congress of the Polish Biochemical Society
Session 3. Proteins — Structure and Function35
L3.5
L3.6
Structural and functional analysis of
EcoG65I restriction-modification system
from clinical isolate Escherichia coli G65
A novel highly thermostable
lysozyme, a PGRP-like amidase from
Thermus bacteriophage Ph2119
Martyna Franczuk, Anna-Karina
Kaczorowska, Tadeusz Kaczorowski
Magdalena Plotka1, Aleksandra Stefanska1, Anna-Karina
Kaczorowska1, Gudmundur O. Hreggvidsson2, Slawomir
Dabrowski3, Janusz M. Bujnicki4, Tadeusz Kaczorowski1
University of Gdańsk, Department of Microbiology, Gdańsk, Poland
e-mail: Martyna Franczuk <[email protected]>
Mobile genetic elements play a crucial role in horizontal
gene transfer (HGT) by facilitating spread of genes coding
for antibiotic resistance, toxins or other virulence factors.
Comparative genomes analysis revealed that HGT is one
of the most important mechanism shaping genetic variability among microorganisms. Restriction-modification (RM)
systems studied in our laboratory affect HGT by modulating the flow of genes among bacteria. There is increasing number of evidences suggesting that this function is
as much important as involvement of the RM systems in
protection of microorganisms against phage infection.
The aim of our study is structural and functional analysis
a RM system EcoG65I of pathogenic strain of Escherichia
coli G65. This clinical isolate was analyzed in our laboratory using PCR as a serotype O26, positive for virulence
determinants eaeA (intimin) and hlyA (hemolysin A) characteristic for verotoxigenic strains of E. coli. The EcoG65I
RM system consists of two enzymatic entities. The first is
restriction endonuclease (R.EcoG65I) which recognizes
specific nucleotide sequence 5’-CTGCAG-3’ and cleaves it
best at 37°C between adenine and guanine residue leaving 3’ four-nucleotide protruding ends. The second constituent is DNA methyltransferase (M.EcoG65I) which
modifies target sequence and thus protects bacterial DNA
against cleavage by the cognate endonuclease. In this respect EcoG65I enzymes are isospecific to PstI RM system
of Providencia stuartii. The genes coding for EcoG65I RM
system have been cloned, overexpressed and their nucleotide sequence was determined. The genes are transcribed in
the same orientation, with the gene coding for M.EcoG65I
(ecoG65IM) preceding the gene coding for R.EcoG65I (ecoG65IR). The two genes overlap by four nt. M.EcoG65I
is predicted to be 489 aa in length (54990,9 Da); the
R.EcoG65I is predicted to be 317 aa (35062,9 Da). The
overall G+C content of these genes is 52,5% (51,6% for
ecoG65IM and 53,4% for ecoG65IR) which is close to E. coli
genomic DNA (50.7%). In order to determine how codon
usage pattern of the EcoG65I RM genes differs from that
of E. coli, the codon adaptation index (CAI) was calculated.
The CAI values obtained (0,27 and 0,25 for the ecoG65IM
and ecoG65IR, respectively) indicate that both genes belong
to the subset of E. coli genes that were most probably acquired by horizontal gene transfer.
Amino-acid sequence of R.EcoG65I shows similarity to
isospecific enzymes: R.BsuBI (69%), R.BbrUIII (44%),
and R.PstI (42%). Whereas aa sequence of M.EcoG65I
shows significant similarity to Eco57I RM enzyme (83%),
M.BsuBI (52%) and M.PstI (42%). The presence and distribution of highly conserved nine aa sequence motifs and a
putative target recognition domain in the enzyme structure
suggest that M.EcoG65I belongs to the exocycling DNA
methyltransferases that modify adenine residues at N6 position.
1University of Gdansk, Department of Microbiology, Gdańsk,
Poland, 2Matis, IcelandicFood and BiotechR&D, Iceland, 3A&A
Biotechnology, Poland, 4International Institute of Molecular and Cell
Biology, Laboratory of Bioinformatics and Protein Engineering, Warsaw,
Poland
e-mail: Magdalena Płotka <[email protected]>
Nucleic acids based technologies now underpin a large and
fast-growing industry, including research, diagnostics and
pharmaceutical production. Thermostable enzymes have
played a key role in this development and polymerases
(DNA and RNA), ligases, nucleases and lysozymes are of
great importance in the research industry today.
Here, we present characterization of highly thermostable
lysozyme from bacteriophage Ph2119 infecting Thermus scotoductus. The phage strain Ph2119 and its host, Th. scotoductus
strain 2119 was isolated from a sample taken at Hrafntinnusker, which is located in the highlands of Iceland, near
the volcano Katla. Its optimum growth temperature is
65°C. The phage 2119 sequencing resulted in 212 contigs
and the bioinformatics analysis revealed the existence of
a 468-bp open reading frame (ORF) encoding a protein
which shows similarity to eukaryotic Peptidoglycan Recognition Proteins (PGRPs), a family of proteins homologous
to T3 and T7 bacteriophages lysozymes.
The tertiary structure of the Ph2119 protein is similar to
Drosophila melanogaster Peptidoglycan Recognition Proteins
(PGRPs): PGRP-Lb, PGRP-Le and PGRP-Sa which all
recognize bacterial peptidoglycan. Additionally, PGRP-Lb
has the ability to directly destroy bacterial peptidoglycan by
hydrolysing the peptidoglycan amide bond. Hence, we decided to explore the possibility that the novel ORF product
possesses the peptidoglycan lytic activity.
The Ph2119 lysozyme gene was synthesized and the recombinant protein was purified from E. coli TUNER
(DE3) harbouring plasmid pET15b [Ph2119lysozyme] using Ni2+ affinity chromatography, yielding 34 mg per 1 litre
of culture. On SDS-PAGE, the purified recombinant protein migrated as a single band with an apparent molecular
mass of 19.7 kDa.
Ph2119 lysozyme bacterial lytic activity was tested. Ph2119
enzyme was mainly active against Gram-negative bacteria
including: extremophile Thermus thermophilus HB8 (100%),
Escherichia coli (34%), Salmonella panama (10%) and Pseudomonas fluorescence (13%). Ph2119 lysozyme showed no activity against Gram-positive bacteria with the exception of
Bacillus cereus (15%). It is noteworthy that Ph2119 lysozyme
can lyse Gram-negative bacteria from without, what is very
unique among lysozymes and perhaps is associated with the
presence of amphipathic characteristics at the C-terminal
end of the protein. Ph2119 lysozyme was found to be
highly thermostable, it maintained approx. 87% of enzyme
activity after 6 h incubation at 95OC. Optimum working pH
was determined as 8.0.
As Ph2119 lysozyme effectively lyses the Thermus thermophilus sp. without the necessity of adding outer membrane
permeabilization agents, it can be used in spheroplasts
preparation, for effective genomic/plasmid DNA isolation
and, most importantly, in enzymes preparations from thermophilic bacteria.
36
Oral presentations
O3.2
O3.1
Nuclear transport receptor CRM1
A novel in silico simulation approach
to identify selective STAT1 inhibitory
compounds to treat atherosclerosis
Anna Kozakiewicz1, Thomas Monecke2,
Piotr Neumann2, Ralf Ficner2
Małgorzata Szeląg1, David Schaller1, Anna Czerwoniec2,
Krzysztof Sikorski1, Joanna Wesoły3, Hans A.R. Bluyssen1
1Faculty
1Department of Human Molecular Genetics, 2Bioinformatics
Laboratory, 3Laboratory of High Throughput Technologies, Institute of
Molecular Biology and Biotechnology, Adam Mickiewicz University in
Poznań, Poznań, Poland
of Chemistry, N. Copernicus University, Poland; 2Institute
of Microbiology and Genetics (GZMB), Georg-August-University
Göttingen, Germany
e-mail: Anna Kozakiewicz <[email protected]>
e-mail: Małgorzata Szeląg <[email protected]>
The exchange of macromolecules between the cell nucleus
and the cytoplasm is tightly restricted by the nuclear pore
complexes (NPCs). Macromolecules such as RNA and
proteins require association with karyopherins called importins to enter the nucleus and exportins to exit. Proteins
that must be imported into the nucleus from the cytoplasm
carry nuclear localization signals (NLSs) that are recognized by importins. Proteins, transfer RNA, and assembled
ribosomal subunits are exported from the nucleus due to
association with exportins, which bind signaling sequences
called nuclear export signals (NESs). The ability of both,
importins and exportins, to transport their cargoes is regulated by the small GTPase Ran. In its GTP bound state,
Ran is capable of binding karyopherins. Importins release
cargo upon binding to RanGTP, while exportins must bind
RanGTP to form a ternary complex with their export cargo. The dominant nucleotide binding state of Ran depends
on whether it is located in the nucleus (RanGTP) or the
cytoplasm (RanGDP) [1].
CRM1is a member of the importin β superfamily of nuclear transport receptors. It mediates nuclear export of numerous cargoes through the recognition of the leucine-rich
nuclear export signal (LR-NES). CRM1 is unusual among
karyopherins in that it has a cargo-binding site on its outer
surface, but this is important for CRM1 to carry a broad
range of cargoes that vary greatly in size and shape, including huge cargoes such as ribosomal subunits. In contrast
to the cargoes that bind to the outer surface of CRM1,
RanGTP binds to the inner surface of CRM1 [2]. CRM1
binds cooperatively to RanGTP and its export cargo, leading to the formation of a trimeric transport complex in
the nucleus [1]. Many amino acids of Ran participate in
the interaction with the CRM1, among others K37 and
K38. To check whether these amino acids are important,
firstly Ran was acetylated on these lysine residues (RanQ69LK37/K38Ac) and then trimeric complexes (CRM1RanQ69LK37/K38Ac-SPN1) were generated.
References:
1. Hutten S et al (2007) Trends Cell Biol 17: 193–201.
2. Monecke T et al (2009) Science 324: 1087–1091.
Signal transducers and activators of transcription (STATs)
comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Crucial
for STAT activation is the Src homology 2 (SH2) domain,
which is highly conserved among the STAT proteins and
interacts with phosphorylated tyrosine motifs for specific
contacts between STATs and receptors and for STAT dimerization. Ligand-induced receptor pTyr binding by the
SH2 domain of STATs allows for phosphorylation of the
protein at the cytoplasmic domain of the receptor and
subsequent formation of transcriptionally active STAThomodimers that move into the nucleus and activate transcription.
STAT1 has been implicated in a number of inflammatory
and autoimmune diseases. We and others previously identified STAT1 as a point of convergence for cross-talk between different pro-inflammatory stimuli in immune and
vascular cells, implicating this transcription factor in development of cardiovascular diseases.
Therefore we decided to search for STAT1-targeting compounds based on computational docking studies exploring
the phosphotyrosine 701/SH2 interaction area of STAT1.
For this purpose, based on existing crystal structures of
human STAT1 and mouse STAT3, we generated new
hSTAT1, hSTAT2 and hSTAT3 protein models. Accordingly three functional cavities could be recognized in the
SH2 domain: pY+0, pY+1 and pY-X that can be targeted
by inhibitors. Our studies of Stattic, known STAT3 inhibitor and Fludarabine, known STAT1 inhibitor revealed that
compounds targeting only pY+0 and pY-X demonstrate
cross-binding specificity between STAT1, STAT2 and
STAT3.
In search of novel specific STAT1-SH2 inhibitors, we performed comparative in silico screens of small-compound
libraries and discovered three lead compounds, which in
ECs in vitro inhibited phosphorylation of hSTAT1 more effectively than phosphorylation of hSTAT3. Subsequent rescreen of drugable compound libraries, using these leads
as templates identified six potential candidates, targeting
pY+0, pY-X and pY+1 cavities of hSTAT1 and not of
hSTAT2 and hSTAT3, making them ideal candidates for
specific STAT1 inhibition.
We developed a novel comparative virtual screening approach to identify specific hSTAT1-SH2 domain inhibitors, that can be used as a novel strategy in treatment of
atherosclerosis.
Acknowledgements:
This research was supported by grants: N N302 016339 (to HB); N N301
073140 (to HB); 0083/IP1/2011/71 (to AC) and 0493/IP1/2011/71 (to
KS) from Polish Ministry of Science and Higher Education and No 128
from the Poznan Supercomputer Center (PCSS) (to MS). This research
was supported in part by PL-Grid Infrastructure.
O3.3
Conversion of 5’-O-monothiophosphate
nucleosides to 5’-O-monophosphate
nucleosides under cellular conditions —
interaction of NMPS with Hint1 enzyme
References:
1. Krakowiak A, Kaczmarek R, Baraniak J, Wieczorek M, Stec WJ (2007)
Chem Comm 2007: 2163–2165.
2. Ozga M, Dolot R, Janicka M, Kaczmarek R, Krakowiak A (2010) J Biol
Chem. 285: 40809–40818.
Agnieszka Krakowiak, Róża Pawłowska,
Beata Rębowska, Wojciech J. Stec
Centre of Molecular and Macromolecular Studies PAS, Department of
Bioorganic Chemistry, Poznań, Poland
e-mail: Agnieszka Krakowiak <[email protected]>
Histidine triad nucleotide binding proteins (Hints) are the
most ancient members of the histidine triad protein superfamily (HIT proteins) of nucleotidyl transferases and
hydrolases. In vitro Hint-1 enzyme exhibits substrate specificity towards many compounds containing different type
of phosphate linkages such as: phosphoramidates (P-N),
phosphates (P-O), phosphorothioates (P-S) and phosphorfluoridates (P-F). We have found that Hint1 can hydrolyze
the P-S bond in adenosine 5’-O-monophosphorothioate
(AMPS) at the rate of 0.2 nmol/min/µg, what is about
40-times faster than the Fhit enzyme does and, moreover,
it is also able to convert other (d)NMPS to (d)NMP under in vitro conditions by mechanism similar to the P–N
bond cleavage in phosphoramidate substrates. [1], [2] Recently, phosphorothioate modification of DNA has been
identified in bacteria so, the ability to hydrolyze P-S bond
in different (d)NMPS by this protein has became more
significant. Furthermore, synthetic oligonucleotide phosphorothioates have been developed as antisense probes
for genomic research and medicinal applications. Their hydrolysis in plasma, kidney, and liver proceeds mainly from
the 3’ end, resulting in the appearance of the nucleoside
5’-monophosphorothioates. The (d)NMPS may exert cytotoxic effects affecting cell proliferation, DNA or RNA
synthesis, and other so far unknown processes, so the
knowledge about their metabolism seems to be of more
wide interest.
Because Hint-1 orthologs were found from prokaryotes to
eukaryotes, we hypothesize that this protein could be the
enzyme responsible for the (d)NMPS metabolism in vivo
and that this process undergoes via their conversion to (d)
NMP.
To prove this assumption, we synthesized non-hydrolysable
phosphorothioate dinucleoside derivative Br5dUO-3’- PSOCH2CH2CH2O-PS5’-OA, which contains 5-bromo-2’deoxyuridine (Br5dU), the residue able to make a covalent bond in nucleic acid-protein complexes by means of
photocrosslinking (at 300 nm). The compound consists of
four diastereoisomers due to chirality at both phosphorus
atoms. We have found that isomer RPRP forms complex
with the recombinant Hint1 the most efficiently. Similar
complexes (probably with Hint1 enzyme) are formed with
proteins present in cellular extracts obtained from A549
cells (Hint1+, Fhit-). In the next step, we have generated
siRNAs directed towards HINT1 mRNA and used them
to transfect A549 cells. Lysates of these cells were used
in experiment with AMPS and the level of desulfuration
was measured. The decreased level of the AMPS desulfuration was observed with cell lysate in which Hint1 was
silenced as compared to the extent of the above reaction
under control condition (cell lysates not transfected with
HINT1-specific siRNAs).
Above results allow for conclusion that intracellular Hint1
enzyme can be the protein responsible for the desulfuration process of nucleoside- 5’-monophosphothioate in vivo.
Undoubtfully, it participates in metabolism of (d)NMPS.
38
O3.4
Posters
Improving PCR specificity by use of
RadA protein from Pyrococcus woesei
P3.1
Aleksandra Stefańska1, Sławomir
Dąbrowski2, Tadeusz Kaczorowski1
Protective effect of Trolox against storage
lesions in red blood cells for transfusion
1University of Gdansk, Department of Microbiology, Gdańsk,
Poland, 2A&A Biotechnology, Gdynia, Poland
Adam Antosik, Natalia Cichon, Magdalena Szejk,
Michal Bijak, Pawel Nowak, Halina M. Zbikowska
e-mail: Aleksandra Stefanska <[email protected]>
Background: Strand exchange catalysts play a pivotal role
in homologous recombination — universal reaction catalyzed by RecA homologues in all domains of life[1]. Our
present studies involve cloning, purification and characterization of a series of such proteins; one of them is RadA
from hyperthermophilic archeon Pyrococcus woesei. The protein of 38.4 kDa was reported to exhibit DNA-dependent
ATPase, D-loop formation and strand displacement activities[2]. Characteristic ability of pairing homologous DNA
sequences with high specificity together with great thermostability of the enzyme encouraged us to test its influence
on PCR specificity. Materials and Methods: Gene encoding RadA was amplified by PCR and cloned into pET15b vector under strong
T7 promoter resulting in a fusion of RadA with N-terminal
His-tag. Expression in BL21(DE3) pRARELys strain provided high yield of soluble protein. Purification was carried
on TALON® affinity resin. Different amounts of purified
RadA together with optimized ATP concentration of 400
µM were tested in model PCR, where a 650 bp long fragment of Human DNA was chosen as a target for amplification. Major problems with efficiency and specificity in
case of standard reaction enabled testing potential positive
effect of RadA activity.
Results and Conclusion: 0.1 µg of RadA per 25 µl of
reaction volume resulted in almost total loss of unspecific
products while the target sequence of 650 bp was amplified
with efficiency much higher than in case of the standard reaction. ATP itself had no positive effect on the specificity,
nor the efficiency of target amplification. Providing positive results of subsequent RadA tests in multiplex PCR, the
protein could be used to solve problems with diagnostic
PCR, where specificity is a crucial issue.
Acknowledgements:
The study is a part of EXGENOMES project financed by UE SEVENTH FRAMEWORK PROGRAMME.
Department of General Biochemistry, Faculty of Biology and
Environmental Protection, University of Lodz, Łódź, Poland
e-mail: Adam Antosik <[email protected]>
The quality of stored red blood cells (RBCs) has improved
with the use of anticoagulant/additive solutions. However,
these storage conditions do not fully preserve viability and
function of RBCs. There is evidence that oxidative damage
to lipids and proteins can contribute to RBC injury during storage which can be manifested by, i.e. enhanced lipid
peroxidation, reduced glutathione (GSH) and haemoglobin
(Hb) concentrations, increased activity of extracellular lactate dehydrogenase (LDH) and haemolysis. Vitamin E is an
essential nutrient for humans and a powerful hydrophobic
antioxidant. It is a scavenger of peroxyl radical that protects membrane lipids and other vital cellular molecules.
The aim of the study was to investigate the effects of
Trolox (a water soluble analogue of vitamin E) on changes
generated in the stored RBCs. The cells were storied at 4oC
in the presence of Trolox (1-125 µM) or without the antioxidant (control). At the 1-, 10- and 20-day of storage the
level of lipid peroxidation (expressed as a concentration
of thiobarbituric acid reactive substances; TBARS), the
activity of extracellular LDH, the concentration of GSH in RBCs as well as % hemolysis in RBC supernatant were
measured. It was shown that addition of Trolox to the RBCs did not
prevent lipid peroxidation during storage. However, Trolox
(125 µM) at the day 20 of storage significantly reduced
extracellular LDH activity and hemolysis as well as it increased the concentration of GSH (by approx. 15% and
20% as well as by above 35% vs. the 20-day control without
the antioxidant, respectively). The results suggest that Trolox would be beneficial as a
cytoprotective and antioxidant agent but other protective
substances are necessary to decrease lipid peroxidation in
stored RBCs.
References:
1. Bianco PR, Tracy RB, Kowalczykowski SC (1998) DNA strand exchange proteins: a biochemical and physical comparison. Front Biosci 3:
D570–603.
2. Komori K et al (2000) Both RadA and RadB are involved in homologous recombination in Pyrococcus furiosus. J Biol Chem 275: 33782–33790.
P3.2
P3.3
Flavonoids as a direct coagulation
factor Xa inhibitors
Mutational analysis of FGF1 and FGF2
to identify binding sites involved in
interaction with new binding partners
Michał Bijak, Adam Antosik, Michał B. Ponczek,
Halina M. Żbikowska, Paweł Nowak
Department of General Biochemistry, Faculty of Biology and
Environmental Protection, University of Łódź, Łódź, Poland
e-mail: Michał Bijak <[email protected]>
Coagulation process consists of a series of zymogens that
can be converted by limited proteolysis to active enzymes
leading to the generation of thrombin, which in turn converts soluble fibrinogen into insoluble fibrin clot. Activated FX (FXa) interacts with its cofactor FVa and forms
on the TF-bearing cell prothrombinase complex which is
responsible for conversion of prothrombin to thrombin.
FXa is a member of S1 family of serine proteases called
chymotrypsine-like with typical for this family active center
(His57, Aps102 and Ser195). Researches carried out in the
last years provided evidence that polyphenol compounds
are able to inhibit activity of many enzymes including serine proteases.
The aim of our study was to examine the effects of the
polyphenol compounds on amidolytic activity of activated
coagulation factor X and to explain this interaction using
bioinformatic analyses.
We observed in our study that from all tested polyphenolic
compounds only polyphenols belonging to flavonoids
group such as: cyanidin, quercetin, procyanidin B2 and silybin had inhibitory effect on FXa amidolytic activity. Bioinformatic analyses performed using Autodock Vina 1.0
showed that cyanidin, quercetin, procyanidin B2 and silybin
bound in the pocket located in vicinity of the FXa active
site and blocked access of substrates to Ser195 and His57.
Similar docking result was obtained for rivaroxaban, FXa
inhibitor using in pharmacotherapy.
The results presented here showed that flavonoids have
potential anticoagulant effect. Controlling of coagulation
factor X activity may prevent uncontrolled thrombin generation process, one of major factors responsible for the
thrombus formation in the blood vessels.
Joanna Bober1, Michał Kostaś2, Justyna Tomala2, Małgorzata
Zakrzewska1, Daniel Krowarsch2, Jacek Otlewski1
1University of Wroclaw, Department of Protein Engineering, Wrocław,
Poland; 2University of Wroclaw, Department of Protein Biotechnology,
Wrocław, Poland
e-mail: Joanna Bober <[email protected]>
Fibroblast growth factor 1 (FGF1) and 2 (FGF2) are the
members of fibroblast growth factor family. They are involved in several biological processes - proliferation, cell
growth, cell survival and morphogenesis by binding to and
activating specific cell surface receptor. Additionally they
possess the ability to translocate across cellular membrane
into the cytosol and nucleus. However, their intracellular
functions remain unknown despite many years of research.
To understand the role of FGF1 and FGF2 inside the cell,
“pull down” experiments followed by mass spectrometry
analysis were performed to identify potential binding partners of these fibroblast growth factors. Based on proteomic approach we identified several proteins, i.e. HSP90,
nucleolin, PCAF and p53. To determine residues of FGF1
and FGF2 involved in the interactions with these proteins,
we designed 18 surface mutations for both growth factors.
These mutations, which disturb putative binding sites, were
selected based on structural information from complexes
of FGFs deposited in PDB and predicted using bioinformatics tools. We used web servers including meta-PPISP
(which predicts the residues that are likely to form the
binding sites), ConSurf (which analyses evolutionary conserved residues) and SWAKK (which detects amino acid
sites that may be under positive selection). Site-directed
mutagenesis, expression and purification of 36 mutational
variants followed by surface plasmon resonance (SPR) experiments were performed. SPR analysis of interactions indicated that some variants exhibit a significant decrease or
total reduction in binding response, suggesting the involvement of selected residues of FGF1 and FGF2 in binding
to cellular partners. Such approach leads from structural
analysis of protein to exploration of its function. Identification of specific binding regions within FGF1 and FGF2
interacting with intracellular partners sheds the light on the
potential role of growth factors in cytoplasm and nucleus.
40
P3.4
P3.5
Expression and purification of
Fibroblast Growth Factor Receptors
1 and 2 (FGFR1 and FGFR2)
Lyn kinase differently regulates
MyD88- and TRIF-dependent signaling
pathways of TLR4 activated by LPS
Aleksandra Borek, Aleksandra SokołowskaWędzina, Jacek Otlewski
Kinga Borzęcka, Agnieszka Płóciennikowska,
Katarzyna Kwiatkowska
University of Wroclaw, Department of Protein Engineering, Wrocław,
Poland
Nencki Institute of Experimental Biology, Department of Cell Biology,
Warsaw, Poland
e-mail: Aleksandra Borek <[email protected]>
e-mail: Kinga Borzęcka <[email protected]>
The FGFR family contains five closely related genes. Four
of them (FGFR1–4) are transmembrane tyrosine kinase
receptors. The family of fibroblast growth factor receptors
(FGFRs) plays an important role in cell growth, survival,
differentiation and angiogenesis. Deregulation of FGFR
signaling has been associated with several developmental
syndromes as well as with many types of cancer.
The first phase of the project comprised expression and
purification of recombinant proteins, which will then serve
as molecular targets for the selection and isolation of antibodies. We expressed extracellular domains of Fibroblast
Growth Factor Receptors: FGFR1 (isoform IIIc) and
FGFR2 (isoform IIIc). FGFR constructs were consisted
of 3 immunoglobulin (Ig)-like domains (D1, D2, D3).
Coding sequences were cloned into vector pLEV113 and
produced by transient gene expression in CHO cell line to
obtain fully glycosylated variants of FGFR1 and FGFR2.
Signal peptide sequences were introduced into the protein
sequences, allowing secretion of proteins into the culture
medium. Proteins were tagged with Fc fragment to allow
both an efficient purification of proteins using affinity
chromatography on Protein A Sepharose and increase their
stability.
FGFR1 and FGFR2 were expressed in CHO cells and
analyzed using gel electrophoresis, Western Blotting and
mass spectrometry. Analysis showed that both receptors
were fully glycosylated. To verify molecular mass of purified proteins we used PNGase F enzyme to deglycosylate
both receptors. Gel electrophoresis and mass spectrometry
confirmed masses of deglycosylated proteins.
These results show that expression in CHO cells and purification using Protein A Sepharose allow to obtain fully glycosylated variants of FGFR1 and FGFR2 that are required
to further studies.
Toll-like receptor 4 (TLR4) is localized in the plasma membrane of macrophages and is activated by lipopolysaccharide (LPS), a component of the outer membrane of Gramnegative bacteria. Under the influence of LPS, TLR4 binds
MyD88/TIRAP adaptor proteins triggering a signaling
pathway that leads to production of pro-inflammatory cytokines. After internalization, TLR4 in endosomes binds
second set of adaptor proteins, TRIF/TRAM, and induces
synthesis of type I interferons and chemokines. Activation of TLR4 by LPS can be dependent on an involvement
of lipid domains, rafts, of the plasma membrane. Therefore, we examined an involvement of raft-residing Lyn
kinase in the two signaling pathways of TLR4. We found
that LPS induced dose- and time-dependent activation of
Lyn kinase, as indicated by phosphorylation of tyrosine
residue 396 of the catalytic domain of Lyn and simultaneous dephosphorylation of inhibitory tyrosine residue 507.
Integrity of rafts was crucial for the activity of Lyn kinase.
To interfere with the activity of Lyn, we obtained a series
of mutated forms of the kinase fused with GFP which
were next overexpressed in RAW264 macrophage-like cells.
The transfection efficiency reached 40% of cell population. An expression of K275R kinase-dead Lyn increased
TRIF-dependent production of chemokine RANTES by
60-75% in cells stimulated with 1-1000 ng/ml LPS. On the
other hand, this mutant form of Lyn did not affect substantially MyD88-dependent production of TNFα. Similar
effects were exerted by R135A Lyn kinase mutated in its
SH2 domain and by doubly mutated Lyn at K275R and
R135A. However, when only SH4 domain of Lyn kinase
was expressed in cells, both TNFα and RANTES production were reduced by 40–50%. The inhibitory effect of
the expression of Lyn-SH4 on TLR4 signaling can be related to exceptionally high transfection efficiency with this
construct, affecting up to 80% of all cells, leading to perturbations in the plasma membrane organization by this
plasma-membrane anchored protein. Taken together our
data indicate that Lyn kinase serves a negative regulator of
TRIF-dependent signaling pathway of TLR4, as interference with the activity of the endogenous kinase increased
RANTES production.
P3.6
P3.7
TrfA structural motifs required for
double stranded DNA binding
Levels of total and High-Molecular
Weight adiponectin in serum of
matched maternal-cord pairs: relations
to fetal anthropometric parameters
Elżbieta Zabrocka1, Katarzyna Bury1, Slawomir
Kubik1, Anna Karłowicz1, Urszula Walkow1, Maria
Moreno2, Rafael Giraldo2, Igor Konieczny1
1University
of Gdansk, Department of Molecular and Cellular Biology,
Gdańsk, Poland; 2Centro de Investigaciones Biologicas, Department of
Chemical and Physical Biology, Spain
e-mail: Katarzyna Bury <[email protected]>
TrfA protein forms specific complexes with DNA in order to initiate RK2 plasmid DNA replication. First, TrfA
monomers bind to direct repeats (iterons) located within
the plasmid origin sequence (oriV) [1]. This event results
in origin opening, melting of dsDNA structure within
oriV AT-rich region [2]. Structure of TrfA nucleoprotein
complex is not yet fully known. Due to high instability of
replication initiators and therefore difficulties with crystallographic analysis, the knowledge about structure of plasmid Rep proteins is limited. Homology modeling revealed
that TrfA (like other plasmid replication initiators) has two
winged-helix domains WH1WH2 at the C-terminal region.
The N-terminal region is disordered in solution and its
structure is not known [3]. The aim of this study was to
identify structural motifs of TrfA responsible for binding
with DNA.
By using in vivo binding assay and SPR analysis we tested
how substitutions located in different regions of TrfA
protein will affect its interaction with dsDNA. The obtained results showed that specific mutations in different
protein regions affect the TrfA nucleoprotein complex
formation. Moreover, we observed compensatory effects
of the analyzed substitutions, which were located outside
of WH1WH2 domains. We concluded that apart from two
WH1WH2 domains, which are typical for plasmid Rep proteins, there is a third DNA binding domain namely DBD3.
We purified both DBD3 and WH1WH2 domains in fusion with SUMO protein and we tested their biochemical properties. Our results demonstrate that for effective
DNA binding all three domains of TrfA are needed. Using approach that combines cross-linking, proteolysis and
mass spectrometry we identify residues in TrfA that are in
contact with dsDNA. We found that motif 339LMCGSDSTRVK349 located in the WH2 domain is directly involved
in iteron binding through the Lysine349.
References:
1. Perri S et al (1991) J Biol Chem 266: 12536–12543.
2. Kolatka K et al (2010) Plasmid 64: 119–134.
3. Pierechod M et al (2009) Protein Sci 18: 637–647.
Magdalena Chełchowska1, Joanna Gajewska1,
Jadwiga Ambroszkiewicz1,Tomasz M.
Maciejewski2, Teresa Laskowska-Klita1
1Institute of Mother and Child, Screening Test Department, Warsaw,
Poland, 2Institute of Mother and Child Department of Obstetrics and
Gynecology, Warsaw, Poland
e-mail: Magdalena Chelchowska <[email protected]>
Objective. Adiponectin as a key regulator of insulin sensitivity could thus be expected to have significant effects
on fetal growth and development. Therefore the aim of
this study was to examine serum High-Molecular Weight
adiponectin (HMW) and total adiponectin concentrations
in pregnant women and umbilical cord blood and their correlations with fetal anthropometric parameters.
Methods. Fortyhealthy pregnant women, attending the Institute of Mother and Child in Warsaw were asked to cooperate in this study. All pregnant volunteers signed a written
informed consent form, approved by the Institute’s Ethical Committee. Venous blood samples were collected from
women in I (median: 12 wk) and III trimester (median: 39
wk). Cord blood was collected immediately post partum
from the umbilical vein after clamping of the cord. Serum
HMW and total adiponectin concentrations were determined by immunoenzymatic method using a commercial
available kits. Statistical analysis was done using the STATISTICA 8.0 and the significance level was set at p<0.05.
Results. Concentrations of adiponectin and HMW isoform in serum of mothers increased during pregnancy and
were 20% higher in third than in the first trimester. Umbilical serum total and HMW adiponectin levels were significantly higher compared with maternal levels at birth (total:
17.98±6.61 µg/ml vs 7.79±3.36 µg/ml; HMW: 13.89±6.67
µg/ml vs 5.12±3.29 µg/ml; p 0.0001), and no correlation
was found between cord and maternal adiponetin levels.
In contrast to the findings in adults cord blood values were
significantly correlated with both birth weight (r=0.62;
p<0.01) and birth length (r=0.5; p0.01).
Conclusion. The levels of total and HMW adiponectin
were higher in umbilical cord serum than in maternal serum. The positive correlations between cord blood values
of this protein and their isoform and birth weight and
length suggest, that rather fetal adiponectin, not maternal
serum adiponectin, may be involved in fetal development
during pregnancy.
References:
Inoue K et al (2010) Int Med 50: 707–712.
Savino F et al (2008) Acta Pediatrica 97: 701–705.
Ong GK et al (2007) Clin Endocrinol 67: 108–114.
Sivan E et al (2004) J Clin Endocrinol Metabol 88: 5656–5660.
42
P3.8
P3.9
Direct determination of phospholipase
D activity by infrared spectroscopy
Homoisocitrate dehydrogenase
from Candida albicans: functional
and structural analysis
Le Duy Do1,2, René Buchet1, Slawomir Pikula2,
Abdelkarim Abousalham1, Saida Mebarek1
1Université de Lyon, France; Université Lyon 1, France; INSA-Lyon,
France; CPE Lyon, France; ICBMS CNRS UMR 5246, France; 2Department
of Biochemistry, Nencki Institute of Experimental Biology, Polish
Academy of Sciences, Poland
e-mail: Le Duy Do <[email protected]>
To determine phospholipase D (PLD) activity an infrared
spectroscopy assay was developed, based on the phosphate
vibrational mode of the phospholipid substrates of the
enzyme such as dimyristoylphophatidylcholine (DMPC),
lysophosphatidylglycerol (lysoPG) dimyristoylethanolamine (DMPE), and lysophosphatidylserine (lysoPS). Characteristic vibrational bands were located at 1230, 1226,
1221 and 1218 cm–1, respectively, and served to monitor
the hydrolysis of phospholipids. The appearance of the
phosphate vibrational band of phosphatidate at 1130 cm–1,
served to monitor the amount of byproduct of the hydrolytic cleavage of phospholipids by PLD. In situ measurements could be performed within less than 20 min, using
2–40 mM DMPC and at least 5–10 ng of S. chromofucus
PLD having specific activity of 30 nmol min–1 µg–1 ( corresponding to 150–300 pmol hydrolysed DMPC per minute)
at pH 8.0 in the presence of 10 mM Ca2+. The feasibility
of the infrared assay using lysoPG, DMPE and lysoPS was
also demonstrated, indicating that various natural phospholipids could be employed as substrates to measure the
PLD activity. Reproducible apparent maximum velocities
(Vmax) were also determined. The direct infrared assay
could be used as a possible screening tool to find specific
PLD inhibitors.
Iwona Gabriel, Marek Wojciechowski, Sławomir Milewski
Gdansk University of Technology, Department of Pharmaceutical
Technology and Biochemistry, Gdańsk, Poland
e-mail: Iwona Gabriel <[email protected]>
Identification and characterization of enzymes which are
essential for fungal survival and/or pathogenicity is crucial for the rational design of antifungal drugs. In the
present communication we report results of our studies
on Candida albicans enzyme — homoisocitrate dehydrogenase (CaHIcDH) — catalyzing biosynthetic reaction from
α-aminoadipate pathway of L-lysine biosynthesis present
only in fungal cells and having no counterparts in mammalian cells.
CaHIcDH encoded by the LYS12 gene was overexpressed
in E. coli cells transformed by pET15b-LYS12 expression
plasmid. It catalyzes the Mg2+ and K+ dependent oxidative
decarboxylation of homoisocitrate (HIc) to alpha-ketoadipate, using NAD+ as a oxidative substrate. The wild-type
enzyme was purified and characterized for its physicochemical properties. kcatwas 0.40 s–1, kcat/KM (NAD+) =
0.44 x 103 M–1s–1 and kcat/KM (HIc) = 8.75 x 103 M–1s–1.
Substrate spectrum analysis demonstrated strict specificity for HIc and no activity with isocitrate and 3-isopropylmalate. It was shown that the possibility of using Ic as a
substrate by T. thermophilus HIcDH was strongly dependent on presence of Arg85 and replacing this residue with
Val prevented isocitrate binding and enhanced activity with
HIc as a substrate. According to the multiple sequence
alignment, Arg85 of TtHIcDH corresponds to Val109
in the CaHIcDH sequence, therefore the strict substrate
specificity of the latter enzyme version is not surprising.
Results obtained by SDS-PAGE, Native-PAGE and sizeexclusion chromatography indicate that the native enzyme
may be composed of four subunits and this assumption
was supported by the results of the homology modelling
of the enzyme molecule. Looking for the molecular basis
of a tetrameric structure by native CaHIcDH — never reported previously for HlcDH from fungal sources — we
identified a characteristic loop, relatively rich in charged residiues (155KKED158) which may participate in intersubunit
interaction. Multiple sequence alignment, amino acid sequence analysis and homology modelling of 3D CaHIcDH
structure revealed the presence of a sequence known as a
isocitrate/isopropylmalate dehydrogenase conserved site:
IPRO19818 (268NLYGDILSDGAAALVGSLGV287) and
the catalytic Lys-Tyr pair (Lys211 and Tyr147), corresponding to Lys206 and Tyr150, shown previously in ScHIcDH
to catalyze the acid-base chemistry of the enzymatic reaction.
P3.10
P3.11
Aggregation test for fragment of
human cystatin C Ala52– Asp65
MOR receptor gene polymorphism (A118G)
in patients with acute pancreatitis
Emilia Iłowska1, Jakub Barciszewski2, Mariusz
Jaskólski2, Aneta Szymańska1
M. Jacuński1, Ł. Jaśkiewicz1, A. Janicka1, K.
Patyra1, M. Matysiewicz2, B. Kocbach2, A.
Cieślińska2, J. Snarska2, E. Kostyra2
1Department of Medicinal Chemistry, Faculty of Chemistry, University
of Gdańsk, Gdańsk, Poland; 2Center for Biocrystallographic Research,
Institute of Bioorganic Chemistry, Polish Academy of Science, Poznań,
Poland
e-mail: Emilia Ilowska <[email protected]>
1The University of Warmia and Mazury in Olsztyn, Faculty of Medical
Sciences, Olsztyn, Poland; 2The University of Warmia and Mazury in
Olsztyn, Faculty of Biology and Biotechnology, Olsztyn, Poland
e-mail: Mateusz Jacuński <[email protected]>
Research carried out on fibrilization process leads to some
new hypotheses concerning the mechanism of this process,
as well as the factors responsible for triggering and progress of fibril formation by a particular protein. One of
them is hypothesis about existence of a specific amino acid
sequences and clearly defined structure which promote
these processes. The presence in proteins structures with
very high propensity for formation of the β-stand conformation, located near the surface and have high ability for
self-association and fibrilizoation called “steric zipper” motif [1, 2]. This motif was suggested by Americans scientists
from prof. Eisenberg’s group. They proved that in amyloidogenic proteins are sequences that promote aggregation
and fibrilization process [1–3].
The aim of this project is to check the applicability of the
steric zipper hypothesis for the analysis and understanding the amyloidogenic propensities of human cystatin C
(hCC). Due to its physical and chemical parameters (good
solubility in aqueous buffers, low molecular mass), cystatin C may be considered as a good model protein in studies aimed at verifying the Eisenberg’s hypothesis. The 3D
profile method and calculated Rosetta algorithm values for
hCC show that in this protein there are two fragments for
which high fibrillization propensity can be expected: the
first N-terminal hairpin fragment encompassing the loop
L1 structure (residues from Ala52 to Asp65) and C-terminal
part of the molecule between Arg93 and Thr116. In this
project we want to do experimental studies to proved this
hipothesis.
Confirmation of existence of the steric zipper motif in the
hCC sequence may have a significant impact on better understanding of the mechanism of self-association, aggregation and fibrillation of this protein.
Acknowledgements:
Work supported by grants: DS/8440-4-0172-13 and and for Young Scientists: 538-8440-B011-13.
References:
1. Goldschmidt L et al (2010) Proc Natl Acad USA 107: 3487–3492.
2. Sawaya MR et al (2007) Nature 447: 453–457.
3. Nelson R et al (2005) Nature 435: 773–778.
Opioid receptors are classified as: mi (µ), kappa (κ), delta
(δ) and sigma (σ). They belong to a large family called G
protein — coupled receptors (GPCR) and they have different significance and physiological functions. µ — receptor (MOR) is important target place for opioid drugs. In
humans, opioid compounds acts on central nervous system showing their wide range of analgesic action, causing
euphoria, sedation, respiratory depression and cough suppression. They also have peripheral effects like constipation. In the process of inflammation, opioids are transported from ganglion cells of dorsal roots to peripheral sensory
nerve fibers. At the same time immunological response
cells, which contain endogenous opioid peptides, gather
themselves within inflammatory altered tissue. Environmental stimulation such as stress, endogenous factors (e.g.
corticotropin-releasing factor, cytokines), can release this
opioids peptides allowing interaction with neuronal opioid
receptors and causing local analgesia.
A large number of point mutations (SNP) have been identified in MOR gene. The most common is A118G polymorphism in exon I — it comes to replacement from adenine
(A) to guanine (G) in position 118. This results in replacing
Asn to Asp in amino acid chain. Nowadays exact molecular
consequences of A118G MOR polymorphism, which may
explain noticed clinical effects, remain unknown. However
it is commonly known that appearance of G allele in polymorphic place of MOR results in decreasing capacity of
the receptors binding. It was shown that people with this
type of polymorphism feel more intensively intoxicating
properties of ethanol. Furthermore people with G allele
confirmed ethanol abuse among their ancestors. Ethanol is
one of the most important risk factors of acute pancreatitis
episode, thus it is possible relationship between MOR gene
polymorphism with acute pancreatitis. The aim of this work was to determine the frequency of
A118G polymorphism of MOR gene in population of
healthy people and people who suffer from pancreatitis as
well as investigation of correlation between this polymorphism and falling for acute pancreatitis.
DNA was isolated from epithelium and blood of healthy
people (control group) and from people with acute pancreatitis (test group). The polymorphism of MOR gene was
determined with PCR — RFLP.
In the whole examined population the frequency of A allele was 0.94 and G allele 0.06. The results indicate the need
for continuation further research on the A118G polymorphism in MOR gene including a wider group of people.
44
P3.12
P3.13
Characterization of leucine-rich repeat
domain containing protein, LRRC45
in ciliate Tetrahymena thermophila
and in mammalian cell lines
Calorimetric evidence of anandamide
— albumin interactions
Katarzyna Jedynak1, Ewa Joachimiak1,2
1Department of Animal Physiology, University of Warsaw, Warsaw,
Poland; 2Laboratory of Physiology of Cell Movements, Nencki Institute
of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland
e-mail: Katarzyna Jedynak <[email protected]>
Leucine-rich repeat (LRR) domain is found in a variety of
proteins and is suggested to be involved in protein-protein
interactions. LRR-containing proteins participate in a wide
range of biological processes, such as DNA repair, transcription, signal transduction, apoptosis, cell adhesion and
immune response. Furthermore, many mutations of proteins from LRR-containing family were shown to be associated with malignant diseases.
The aim of this studies was determination of function and
localization of LRRC45 protein in Tetrahymena thermophila
and in mammalian cell lines.
We choosed model organisms Tetrahymena thermophila, murine cell line NIH 3T3 and human cell line A431 to investigate the impact of overexpression and silencing of protein
LRRC45 on ultrastructure, and did biochemical and phenotypic studies.
We analyzed Tetrahymena thermophila genome and available
proteomes and identified pellicular LRR-containing protein. Our initial studies showed that the identified protein
is a novel cytoskeletal proteins. In T. thermophila, these LRR
protein localize to the cortical cytoskeleton near the basal
bodies but the function and linking with one of the cell
compartments remain unknown. When the overexpression
is on high level protein fused with GFP forms structures
resembling vesicles of unknown origin. Overexpression
of GFP-LRRC45 causes acceleration of Tetrahymena thermophila cell division rate as opposed to overexpression of
LRRC45 without tag that reduces multiplication rate. Control and mutant cells were grown in medium supplemented
with CdCl2. The cells density was determined every three
hours.
Tetrahymena LRRC45 protein has single human homologue,
HsLRRC45. We demonstrated that in cells overexpressing
HsLRRC45 fused with GFP on C- or N- terminus, the recombinant protein localized to juxtanuclear vesicular-like
structures. The intracellular localization resembles localization of activated Rab5, caveolin-1 or the early endosome
marker, EEA-1. The origin of these vesicles was determined by testing their putative colocalization with markers
specific for different membranous structures like transport
vesicles, lysosomes, autophagosomes. We demonstrated
that there was incomplete colocalization with cis-Golgi
marker GM130 and overexpressed and nativ HsLRRC45
protein. Furthermore the level of HsLRRC45 protein
depends on cell line and is inversely proportional to the
GM130 protein level. Finally, we identified the potential
protein partners of the studied Tetrahymena protein and
it’s mammalian homologue using immunoprecypitation,
among which both basal bodies proteins, vesicular trafficking and microtubular cytoskeleton associated proteins were
present.
Overall, our results suggest that LRR domain containing
novel protein probably participate in vesicular trafficking,
probably between Golgi apparatus and endoplasmic reticulum.
Marek Kaczyński1, Krzysztof Bilmin2, Magdalena
Przybyło1, Paweł Grieb2, Marek Langner1
1Institute of Biomedical Engineering and Instrumentation, Wrocław
University of Technology, Wrocław, Poland; 2Department of
Experimental Pharmacology, Polish Academy of Sciences Medical
Research Center, Poland
e-mail: Marek Kaczynski <[email protected]>
Anandamide, (arachidonylethanolamide, AEA) is an endogenous cannabinoid neurotransmitter. Since its discovery in 1992, several other functions have been reported
and especially interesting is its anti-cancer activity. However if one would like to introduce the molecule into the
organism, several problems arise. Because of its chemical
structure, AEA is poorly soluble in water and susceptible to
enzymatic degradation. It is also highly possible that arachidonic residue of anandamide will associate with proteins
that have the ability to bind fatty acids, e.g. albumins, therefore its pharmacokinetics may be altered.
Albumin is the most abundant plasma protein (35–50 g/L
human serum). It is a monomeric, multi-domain, nonglycosylated, negatively charged plasma protein, with numerous
functions important for maintaining the body homeostasis.
HSA is the main determinant of plasma oncotic pressure
and the main modulator of fluid distribution between
body compartments. It is characterized by an extraordinary
ligand-binding capacity, being a carrier for many endogenous and exogenous compounds. Due to its high affinity
to long-chain fatty acids, albumin plays an important role in
their transport and in the modulation of their metabolism.
The question of possible anandamide – albumin interactions arisen in the course of the in vitro studies in which
the antiproliferative properties of AEA were studied. The
HeLa cells growth have been halted only when anandamide
had been introduced in serum free medium. In order to
verify the hypothesis that the studied compound interacts
with albumin, the isothermal titration calorimetry studies
have been performed to determine thermodynamic parameters of anandamide-albumin interactions.
Because of the compound low solubility the liposomal formulation of AEA has been designed so the binding experiment could be successfully performed.
Results obtained with the isothermal titration calorimetry
show distinct difference between the binding parameters
of anandamide interacting with albumin when the fatty
acids have been or have not been present. The obtained
results and developed methodology can be used in the process of the developing new formulations for anti-cancer
cannabinoid drugs.
Acknowledgements:
Presented works were co-financed by the European Union as part of
the European Social Fund and Institute of Biomedical Engineering and
Measurements, Wrocław Technical University.
P3.14
P3.15
Influence of polisiloxanes on the
stability and activity of collagen
Lon protease — mechanism of
proteins degradation on DNA Leszek Kadziński1, Zyta Banecka-Majkutewicz2, Magdalena
Prokopowicz3, Jerzy Łukasiak4, Bogdan Banecki1
Katarzyna Wegrzyn, Marta Gross, Anna
Karlowicz, Igor Konieczny
1Intercollegiate Faculty of Biotechnology University of Gdańsk Medical University of Gdańsk, Department of Molecular and Cellular
Biology, Gdańsk, Poland; 2Medical University of Gdańsk, Department of
Neurology, Gdańsk, Poland; 3Medical University of Gdańsk, Department
of Physical Chemistry, Gdańsk, Poland; 4Gdansk Management College,
Gdańsk, Poland
Intercollegiate Faculty of Biotechnology UG-MUG, Department of
Molecular and Cellular Biology, Gdańsk, Poland
e-mail: Leszek Kadziński <[email protected]>
Silicone polymers are assumed to be chemically stable in
living systems. Therefore they have been used in the medical field for many years as biocompatible biomaterials ranging from intraocular lenses to breast implants. Silicone fluids are widely used as a surgical tamponade in severe cases
of vitreoretinal pathology and as an compounds of bioactive organic-inorganic materials and drug delivery systems.
An inherent problem associated with implanted prostheses
is their propensity to be coated in host proteins shortly after implantation. Some of research works, specifically those
that focused on the implanted silicone prostheses, suggest
that silicone is adjuvant to the human immune system and
may induce conformational changes and aggregation of
the proteins. Furthermore patients with silicone prosthesis display a variety of nonspecific syndromes that usually
disappear after the removal of the implant. In this work
we investigated the effect of cyclic and linear siloxanes on
the structure and function of collagen — the major structural protein in human body. Using the FITC-labelled collagen we have shown protein-siloxanes complexes. We have
demonstrated the influence of the protein-siloxanes interactions on the collagen fibril formation — the main function of collagen molecules. We have also determined that
collagen-siloxanes interactions do not change the conformation of triple helical collagen molecules.
e-mail: Anna Karlowicz <[email protected]>
Lon belongs to the family of ATP-dependent proteases
involved in protein quality control. Bacterial Lon is considered to be the most important factor involved in the
degradation of misfolded proteins. This protease consists
of three domains: the highly variable N-terminal domain,
the central AAA+ ATPase domain containing nucleotide
binding site, and the C-terminal peptidase domain. It forms
barrel-like shaped oligomers with a central cavity within the
macromolecular structure. To date, crystallographic data
are available for full length Lon from Bacillus subtilis and
Thermococcus onnurineus (Duman & Lowe, 2010; Cha et al.,
2010) and only fragments of enzymes from a few other
organisms (including Escherichia coli) (Li et al., 2010; Botos et
al., 2004; Botos et al., 2005). It has been shown that Lon
protease possesses the ability to bind DNA however, no
requirements considering protein structure have been described so far (Zehnbauer et al., 1981). Moreover, our data
suggest that nucleoprotein formation by Lon is required
for its proteolytic activity. We propose quaternary structure of the hexameric E. coli
Lon protease predicted on the basis of data on crystallized fragments of Lon and homology modeling of the
remaining parts of the protein. The analysis of our Lon
structure prediction model revealed potential regions and
residues possibly involved in DNA interaction. Utilizing
site-directed mutagenesis we introduced uncharged or
negatively charged substitutions and created series of Lon
protein mutants. After purification step we tested them using in vitro methods for DNA binding and their activities towards substrates that specifically bind or do not bind DNA.
Our results allowed us to indicate amino acid residues of
Lon engaged in DNA interaction. Moreover, Lon mutants
lacking DNA-binding ability were not able to degrade substrates which specifically bind nucleic acid. Our results confirm crucial role of nucleoprotein complex formation by
Lon for its proteolysis of substrates interacting with DNA.
References:
Botos I et al (2004) J Biol Chem 279: 8140–8148.
Botos I et al (2004) J Struct Biol 146: 113–122.
Cha SS et al (2010) EMBO J 29: 3520–3530.
Duman RE & Lowe J (2010) J Mol Biol 401: 653–670.
Li M et al (2010) Acta Cryst 66: 865–873.
Zehnbauer BA et al (1981) Proc Natl Acad Sci USA 78: 2043–2047.
46
P3.16
P3.17
Desensitization of glucosamine-6phosphate to inhibition by UDP-GlcNAc
Karolina Kwiatkowska, Agnieszka
Napiorkowska, Slawomir Milewski
Analysis of Aβ1–40 peptide oligomers
using ion mobility separation coupled
with ms and factors influencing the
peptide structure compaction
Gdansk University of Technology, Department of Pharmaceutical
Technology & Biochemistry 11/12 Narutowicza St., 80-233 Gdansk,
Poland
Marcin Kłoniecki1, Agnieszka Jabłonowska1, Jarosław
Poznański1, James Langridge2, Kevin Giles2, Nick
Tomczyk2, Chris Hughes2, Michał Dadlez1,3
e-mail: Karolina Kwiatkowska <[email protected]>
Glucosamine-6-phosphate (GlcN-6-P) synthase, known
also as L-Glutamine: D-fructose-6-phosphate amidotransferase, catalyzes the first committed step in the pathway
leading to the ultimate formation of UDP-GlcNAc. The final product of this pathway is an activated precursor of numerous macromolecules containing amino sugars, including chitin and mannproteins in fungi, peptidoglycan and
lipopolysaccharides in bacteria, and glycoproteins in mammals. The reaction catalyzed by the enzyme is conversion
of fructose-6-phosphate into glucosamine-6-phosphate
with L-glutamine as a nitrogen donor.
The important differences between prokaryotic and eukaryotic forms of the enzyme include a substantially larger
size of the latter and sensitivity of the latter but not the
former to the feedback inhibition by uridine 5’-diphosphoN-acetyl-D-glucosamine (UDP-GlcNAc). The enzyme has
been proposed as a promising target in antifungal chemotherapy as well as for the treatment of insulin-independent
diabetes mellitus.
Our studies have been focused on GlcN-6-P synthase
from human pathogenic fungi Candida albicans coded by
the GFA1 gene. We were able to demonstrate a crystal
structure of the isomerase domain (ISOM) of C. albicans GlcN-6-P synthase in complex with UDP-GlcNAc
[2]. Now we report results of our further studies involving the site-directed mutagenesis of GFA1, leading to the
following substitutions of amino acid residues located at
the putative UDP-GlcNAc binding site in the gene products: Gly474Leu, Thr487Ile, Gly490Leu and His492Phe.
Muteins obtaned at consecutive steps of mutagenesis were
isolated and characterized. The final product of mutagenesis was completely insensitive to inhibition by UDP-GlcNAc, while sensitivities of the intermediate products were
diminished. Conclusions concerning the probable role of
UDP-GlcNAc binding for enzyme structure and catalytic
properties are presented.
References:
[1] Milewski S (2002) Biochim Biophys Acta 1597: 173–192.
[2] Raczynska J et al (2007) J Mol Biol 372: 672–688.
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Department of Biophysics, Warsaw, Poland; 2Waters Corporation,
Atlas Park, Simonsway, Manchester, UK; 3Institute of Genetics and
Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland
e-mail: Marcin Kłoniecki <[email protected]>
Alzheimer’s disease (AD) poses a serious challenge on social care systems worldwide and this justifies intense studies on mechanisms underlying AD pathology and on the
nature of the neurotoxic agent. Several pieces of evidence
point to a causative link between the soluble and non-monomeric forms of Aβ peptide, derived by proteolysis of APP
protein, and AD, founding the basis for the revised amyloid cascade hypothesis. It has been argued that the level
of cognitive disability correlates better with the pool of Ab
oligomers, rather than with aggregated amyloid deposits. It
became clear that different Aβ species, also extracted from
AD patient’s tissues, exert neuro/synaptotoxicity in a wide
range of experimental setups. Thus, “oligomer hypothesis”
is central for AD studies.
To gain insight into the Aβ1–40 oligomer structure we have
applied Ion Mobility separation (IMS) coupled with MS. It
allowed us to determine the mobility of Aβ1–40 oligomer
ions and, on the basis of the mobility, calculate the relative collisional cross-section for each oligomeric form observed. For several oligomers, at least two different species
of different Ω values were detected, indicating the presence of at least two families of conformers: compact and
extended. When incubated at room temperature for 24 h
the drift time profile of several oligomeric forms changes
in general with the shift towards more compact forms. The
strongest changes were observed from trimers to pentamers showing that a compact structure becomes dominant
with time. Similar shift was observed for higher oligomers, hexamers and heptamers. This, however, does not hold
for dimeric signals in which the mutual population of both
forms barely changes after 24 h. In general, whenever in
a given charge state of oligomers larger than dimers both
compact and extended forms are present in the spectra of
freshly dissolved peptide, the fraction of compact species
increases with time. These oligomers equilibrate towards
the domination of the most compact species at longer
times. Interestingly similar effect can be obtained just after dissolution of the Aβ peptide if metals are added. An
addition of copper or zinc caused that the drift time profile of several metal-oligomeric complexes shifts towards
more compact forms. Experimentally observed behavior
of higher Aβ species by IMS in gas phase allowed us to
construct a set of molecular models of Aβ oligomer structure that provided a very good correlation between the experimental and theoretical Ω values for both compact and
extended forms. Moreover, in case of the open form, the
model explains the flexibility that enables the formation of
potentially neurotoxic channel-like structures that are able
to dysregulate calcium ion flux at the synapses.
P3.18
P3.19
The structural studies of β-lactoglobulins
from the whey fraction of milk
The molecular characteristics of the
nuclease from extreme psychrophilic
bacterium Psychromonas ingrahamii 37
Joanna Loch, Mateusz Czub, Mira Ludwikowska,
Sylwia Świątek, Krzysztof Lewiński
Faculty of Chemistry, Jagiellonian University, Kraków, Poland
e-mail: Joanna Loch <[email protected]>
β-Lactoglobulin (LG) is a protein present in milk of several species as e.g. cow, goat, sheep, and pig. The core element of LG structure is an antiparallel β-barrel surrounded
by flexible loops that is a primary binding site for ligands.
The binding of ligand in β-barrel requires conformational
changes of EF loop in the process called Tanford transition. The most widely studied bovine lactoglobulin (BLG)
can bind hydrophobic ligands but the selectivity of binding
is very low. To increase affinity to selected compounds, we
plan to re-engenieer the protein. Such modification will allow to utilize it in the future as a transporter of bioactive
compounds with potential applications in medicine.
To characterize the mechanism of ligand binding, structural studies on bovine and goat (GLG) protein have been
performed. BLG and GLG have different amino acids
at six positions L1I, D53N, D130K, S150A, E158G and
I162V. Such substitutions seem to affect not only the physic-chemical properties of protein but also its alergenicity
and ability of ligand binding, although all substitutions are
outside the binding site. BLG and GLG have been isolated
from milk by salting-out and ion-exchange chromatography. Proteins have been crystallized using vapour-diffusion
method in hanging drop set-up. Structures have been
solved by molecular replacement.
Three crystal forms (P3121, P21212 and P3221) of GLG
have been obtained, the BLG was crystallized only in P3221
and C2221 space group. The comparison of BLG and GLG
structures suggests that differences in amino acids composition do not affect the conformation of main chain. The
GLG structures revealed that conformational changes accompanying Tanford transition include not only EF loop
but also the GH loop. The interactions between Asn90
- Ser110 and Glu89 - Ser116 seem to play main role in this
phenomenon. Comparison of BLG and GLG complexes
with ligands revealed the position of the ligand in the binding site is slightly different in BLG and GLG. Crystallization experiments showed that crystals of BLG complexes
of with ligands can be obtained at pH 7.5 and higher but
crystallization of similar GLG complexes requires the pH
8.5. In the GLG structure secondary ligand binding site has
been indentified in the region of GH loop.
Natalia Maciejewska1,2, Aneta Szymańska1,
Emilia Iłowska1, Marcin Olszewski2
1Department of Medicinal Chemistry, University of Gdańsk, Gdańsk,
Poland; 2Department of Microbiology, Gdańsk University of
Technology, Gdańsk, Poland
e-mail: Natalia Maciejewska <[email protected]>
Information regarding psychrophilic organism proteins
is exceptionally limited, particularly given their proliferation; approximately 85% of the earth’s biosphere is psychrosphere, with a temperature of below 5ºC. This is, in
part, owing to the difficulties involved in their acquisition.
Scarcely more than a dozen protein structures of psychrophilic origin are known at present. Such proteins display
high activity at low temperatures. One of the most desirable psychrophilic microbiological products are enzymes,
in other words, nucleases.
Nucleases are important molecular tools belonging to the
group of hydrolase-degrading nucleic acids. Nucleases are
important molecular tools belonging to the group of hydrolase-degrading nucleic acids. Depending on the kind of
substrate digested, it is possible to distinguish deoxyribonucleases, which hydrolyze deoxyribonucleic acids, and ribonucleases, which degrade ribonucleic acids. By the same token, depending on the activity site, we are able distinguish
endonucleases, which hydrolyze intermolecular phosphodiester bonds in a polynucleotide chain, as well as exonucleases, which cleave to the terminal nucleotides of the chain.
We report the identification and characterization of the nuclease from extreme psychrophilic bacterium Psychromonas
ingrahamii 37 that grows expotentially at –12°C and may
well grow at even lower temperatures. Psychrophilic microorganisms, living in extremely cold environments, produce
enzymes which are adapted to performing reactions at a
low temperature. This is extremely important in the case
of nucleases, as it will allow their utilization in the reaction
for the elimination of nucleic acids from thermosensitive
preparations.
Acknowledgement:
The work was supported by grant NCN 2011/01/N/NZ1/01569.
The publication is financed from European Social Fund in as a part of
the project "Educators for the elite - integrated training program for
PhD students, post-docs and professors as academic teachers at University of Gdansk" within the framework of Human Capital Operational
Programme, Action 4.1.1, Improving the quality of educational offer of
tertiary education institutions.
48
P3.20
P3.21
Identification and characterization of class
III UvrA protein from Stigmatella aurantiaca
Renalase levels and activity in blood
and urine of healthy adult volunteers
Marta Marszałkowska1, Magdalena Bil2,
Łukasz Kreft1, Marcin Olszewski1
Natalia Matoszka1, Katarzyna Siewierska2,
Magda Wiśniewska3, Barbara Dołęgowska1
1Gdańsk University of Technology, Department of Microbiology,
Chemical Faculty, Gdańsk, Poland; 2Katholieke Universiteit Leuven,
Department of Biology, Research group of Insect Physiology and
Molecular Ethology, Leuven, Belgium
1Department od Medical Analytics, Pomeranian Medical University,
Szczecin, Poland; 2Students Learned Society, Department of Medical
Analytics, Pomeranian Medical University, Szczecin, Poland; 3Clinic
of Nephrology, Transplantology and Internal Diseases, Pomeranian
Medical University, Szczecin, Poland
e-mail: Marta Marszałkowska <[email protected]>
e-mail: Natalia Matoszka <[email protected]>
UvrA protein is a part of UvrABC repair system, which
catalyzes the recognition and processing of DNA lesions.
This protein is one of the key components of NER repair system pathway in prokaryotes. In last years, several
works have been published about discovering additional
uvrA genes in chromosomes of some prokaryotic species
(Xanthomonas axonopodis, Pseudomonas putida or Deinococcus radiodurans).
We investigated sequences of over 2 400 bacterial genomes
and as a result we found 130 examples of bacteria containing uvrA2 genes. Sequence analyses conducted on these
homologues revealed that previously established division
of uvrA genes was incorrect. Therefore we created a new
division based on our uvrA sequence’s base and current
knowledge about UvrA proteins. This division consist of
four main uvrA classes and five subclasses, among which
only class I genes are well characterized.
In order to investigate the problem of UvrA homologues
and their role in bacteria, we decided to conduct expression and characterize uvrA2 gene product from mesophilic,
aerobic bacteria — Stigmatella aurantiaca. As a next step we
are planning to obtain its crystal structures and examined
its function using mutagenesis techniques.
References:
Goosen N et al (2008) DNA Repair 7: 353–379.
Jaciuk M et al (2012) Nat Struct Mol Biol 18: 191–197.
Pakotiprapha D et al (2008) Moll Cell 29: 122–133.
Shen CH et al (2007) Mol Genet Genomics 277: 149–60.
Tanaka M et al (2005) J Bacteriol 187: 3693–3697.
Tark M et al (2008) DNA Repair 7: 20–30.
Timmins J et al (2009) Cell 17: 547–58.
Truglio JJ et al (2006) Chem Rev 106: 233–52.
Background: Renalase is a novel, soluble flavoenzyme
with potential ability to oxidize biogenic amines. It is expressed mainly in kidney and heart, but also, in a lesser
extent, in skeletal muscles, liver, small intestine, brain or
gonads, and is secreted into the blood and urine. When
discovered (2005), it was described as third monoaminoxidase – MAO-C; the most recent direct research of renalase
activity (2012), however, revealed a different way of action than known monoaminooxidases, but did not exclude
the potential catecholamines-degrading properties, and
cardioprotective effects confirmed in many other studies.
Here, we strongly suggest that renalase metabolizes biogenic amines — especially adrenaline — to corresponding
aminochromes, what is a confirmation of the mentioned
above research made by the renalase discoverers and the
leading renalase researchers — G.V. Desir and co-workers.
Nevertheless, there is still a huge problem to resolve —
how renalase contributes to lowering blood pressure and
preventing many cardiovascular disorders, while producing
toxic aminochromes?
Aim of the study: Aminochromes are natural products
of catecholamines oxidation, including autooxidation, with
potential toxic properties. They are present at very low
concentrations in blood and urine, and quickly neutralised.
Our aim was to test, develop, and in further perspective standarize, the method of measuring renalases activity in
blood and urine of healthy volunteers, assuming that renalase indeed oxidizes catecholamines to aminochromes.
Materials and methods: Concentration of renalase in
urine and blood serum of healthy volunteers was measured
using commercial ELISA kit. All the kinetic parameters,
including NADH+H+ oxidation, production of hydrogen
peroxide and formation of adrenochrome from adrenaline
were measured using spectrophotometric methods. General blood and urine biochemical analyses were carried out.
Results: Amount of adrenochrome, produced in one minute from 1ng of renalase was almost the same in blood and
urine and depended mainly on the time lapse of reaction. Concentration, and therefore activity of renalase – measured by the amount of formed adrenochrome, was about
two times higher in urine than in blood serum.
Conclusions: Obtained results suggest, that adrenochrome
is in fact the product of renalase enzymatic activity in presence of adrenaline and depends on renalase concentration,
therefore amount of adrenochrome formed in presence of
renalase in certain conditions can be considered as constant
parameter. 48th Congress of the Polish Biochemical Society
P3.22
P3.23
Multi-method approach to structure
and function of the mRNA 5’ capbinding proteins: eIF4E and PARN
Specific interactions between the
amyloid precursor and cholesterol
in a lipid bilayer by means of
molecular dynamics simulations
Anna Niedźwiecka1,2, Małgorzata Lekka3, Remigiusz Worch1,
Per Nilsson4, Edward Darżynkiewicz2, Anders Virtanen4
1Institute of Physisc PAS, Laboratory of Biological Physics, Warsaw,
Poland; 2University of Warsaw, Faculty of Physics, Division of Biophysics,
Warsaw, Poland; 3Institute of Nuclear Physics PAS, Kraków, Poland;
4Uppsala University, Department of Cell and Molecular Biology,
Uppsala, Sweden
e-mail: Anna Niedźwiecka <[email protected]>
A biophysical bases of molecular mechanisms underlying
the recognition of the mRNA 5’ terminal structure called
“cap” by proteins is crucial both for understanding of the
complex process of regulation of eukaryotic gene expression at the levels of translation and mRNA surveillance, as
well as for putative drug design.
Recognition of the 5’ cap by the eukaryotic initiation factor
4E (eIF4E) is the rate limiting step of protein biosynthesis, while poly(A)-specific ribonuclease (PARN) is a 5’ capdependent enzyme that plays a key role in 3’ deadenylation,
is involved in nonsense-mediated mRNA decay, and also in
regulation of cytoplasmic polyadenylation. The goal of the
studies was to find structural requirements for the affinity
of the cap-binding proteins to the cap [1], thermodynamic
driving forces [2, 3] and kinetic characteristics of the intermolecular recognition, as well as to gain an insight into
the structure and structural dynamics, that are biologically
relevant.
We have established a precise method of the protein-ligand
binding constants determination [4] and found that eIF4E
exploits conformational changes to provide tight binding
of the cap and the synergy of interactions with eIF4G/4EBP1 [1-3], while PARN is the only one among 3’ exoribonucleases which interacts with the 5’ mRNA terminal
structure [5] to provide the processivity of deadenylation.
PARN is thus the minimal protein context to bind two
mRNA termini concurrently [6]. eIF4E and PARN share
similar structural cap-binding motif (Trp-m7G) but they
have different thermodynamic and kinetic binding properties that correlate with the biological functions of these
proteins. We have also visualized single PARN molecules
by Atomic Force Microscopy in liquid [7] that provided
mesoscopic structural description complementary to the
protein fragments known from cryslallography.
Łukasz Nierzwicki, Jacek Czub
Gdansk University of Technology, Department of Physical Chemistry,
Gdańsk, Poland
e-mail: Łukasz Nierzwicki <[email protected]>
C99 is a transmembrane protein, which was identified as
the precursor of β-amyloid. It is generated from the amyloid precursor protein (APP) by β-secretase. Cleaving of
C99 by γ-secretase in a cell membrane relases β-amyloid
polypeptides, which are associated with Alzheimer disease (AD). According to the work by Barrett et al. [1], C99
when present in a lipid environment, can specifically bind
a cholesterol (CHL) molecule. This might induce cleaving
by γ-secretase either by increasing the affinity of C99 to
lipid rafts in which γ-secretase occurs or by direct participation of cholesterol in the cleaving process. Investigating
such interactions may reveal a new potential ways of drugs
design, as it was proposed that preventing of the binding
between C99 and cholesterol may interfere with. Here we
used molecular dynamics (MD) simulations to investigate
the association between C99 and cholesterol in a lipid bilayer at molecular level. The overall orientation of C99
in a lipid bilayer of different cholesterol content as well
as the ordering effect of the protein on membrane lipids
was investigated by means of equilibrium MD simulations.
The Gibbs free energy profiles for binding cholesterol to
the C99 protein was determined by the umbrella sampling
method. The obtained profiles confirmed the specific binding between C99 and cholesterol and allowed us to propose the structure of the complexes. Two minima found
in the free energy profiles correspond to the two possible
low-energetic structures of the CHL-C99 complex. Using
the aquired MD data, we analyzed molecular driving forces
governing the specific interactions between C99 and cholesterol. To validate our results, MD simlations were also
performed for the E693A mutant which was found to bind
cholesterol much less efectively. Based on these simulations,
we propose the possible molecular mechanism explaining
the observed decrease in affinity of C99 to cholesterol.
Reference:
1. Barrett P et al (2012) Science 336: 1168–1170.
Acknowledgements:
This work was partially performed in the laboratories founded by NanoFun POIG.02.02.00-00-025/09
References:
1. Niedzwiecka A et al (2002) J Mol Biol 319: 615–635.
2. Niedzwiecka A et al (2002) Biochemistry 41: 12140–12148.
3. Niedzwiecka A et al (2004) Biochemistry 43: 13305_13317.
4. Niedzwiecka A et al (2007) Methods Enzymol 430: 209–245.
5. Nilsson P et al (2007) J Biol Chem 282: 32902–32911.
6. Wu M et al (2009) Structure 17: 276–286.
7. Niedzwiecka A et al (2011) Biophys Chem 158: 141–149.
50
P3.24
P3.25
Characterization of single-stranded
DNA-binding proteins from the
psychrophilic bacteria
Effect of various protein receptors
on the catalytic activity of
rattelsnake phospholipase A2:
spectrofluorimetric studies
Marcin Olszewski, Marta Nowak, Marta Śpibida
Gdańsk University of Technology, Department of Microbiology, Gdańsk,
Poland
e-mail: Marcin Olszewski <[email protected]>
Single-stranded DNA-binding proteins (SSBs) are indispensable elements in the cells of all living organisms. SSB
proteins interact with ssDNA in sequence in an independent manner, preventing them from forming secondary
structures and from degradation by nucleases. In this way,
SSB-binding proteins participate in all processes involving
ssDNA, such as replication, repair and recombination. Although there are differences in amino acid sequences, SSBs
have a high-conservative domain, the oligonucleotide/
oligosaccharide–binding fold, referred to as the OB-fold,
which is responsible for binding with ssDNA. In the single-stranded DNA-binding proteins described so far, four
OB-fold domains form an active protein. These proteins
have also the ability to bind RNA and are present in all
three branches of live organisms and in viruses. The cooperative binding of single-strand DNA and RNA which
is a property of SSBs has led to their being used as tools
in molecular biology methods and analytics. Thermostable
proteins are particularly useful in this respect. To date, only
a few thermostable SSB proteins with these valuable applications have been identified and, as yet, no studies have
been undertaken into single-strand binding proteins from
cold-adapted organisms and their applications.
We report the characterization of single-stranded DNAbinding proteins from the psychrophilic bacteria Desulfotalea psychrophila (DpsSSB), Flavobacterium psychrophilum
(FpsSSB), Psychrobacter arcticus (ParSSB), Psychrobacter cryohalolentis (PcrSSB), Psychromonas ingrahamii (PinSSB), Photobacterium profundum (PprSSB) and Psychroflexus torquis (PtoSSB). The
proteins show a high differential within molecular mass of
their monomers and length of amino acid sequences. The
high level of identity and homology in respect of EcoSSB
relates to the OB-fold and some of the last amino acid
residues. The fluorescence titrations indicated that the
ssDNA-binding site size is about 32 nucleotides long and
is salt independent. The DpsSSB, FpsSSB, ParSSB, PcrSSB,
PinSSB, PprSSB and PtoSSB cause the destabilization of duplex DNA, as expected. The greatest decrease of duplex
DNA melting temperature was observed in the presence
of PtoSSB (17°C). The investigated SSBs possess relatively
high thermostability for the proteins derived from coldadapted bacteria.
Maciej Ostrowski1,2, Pierre-Jean Corringer1, Grazyna Faure1
1Institut Pasteur, Department of Neuroscience, Unite RecepteursCanaux, France; 2Nicolaus Copernicus University, Department of
Biochemistry, Poland
e-mail: Maciej Ostrowski <[email protected]>
The secreted phospholipases A2 of IIA group (PLA2) are
small, disulfide bridge-rich proteins which were found in
human synovial fluid and snake venoms. These enzymes
catalyze hydrolysis of glycero-phosholipids at sn-2 position,
releasing fatty acids and lysophospholipids. The group IIA
PLA2s interact with various protein targets, in agreement
with their multifunctional nature (Faure & Saul, 2012).
The biological effects of PLA2 are determined by their
enzymatic activity (regulation of plasma membrane lipid
composition, production of an arachidonic acid for eicosanoids biosynthesis, etc.) or/and by specific high-affinity
interaction with various pre- and post-synaptic receptors
and human coagulation factor Xa. Two novel specific targets of PLA2 were recently identified: the Cystic Fibrosis
Transmembrane Conductance Regulator (CFTR) (Faure et
al, Edelman, submitted) and bacterial pentameric protongated ion channel (GLIC) (Faure et al., Corringer, manuscript in preparation). To better understand the mechanism
of interaction between protein targets and PLA2s, in the
present study we investigated the effect of direct binding of three different proteins (human coagulation factor
Xa, nucleotide binding domain 1 of CFTR, and GLIC) on
the enzymatic activity of PLA2. PLA2 activity was determined by spectrofluorimetric method using pyrene-labelled
glycero-phospholipid as substrate. Kinetic parameters
(Km,Vmax,kcat, kcat/ Km) of PLA2 were determined, showing different regulatory effects of FXa, NBD1-CFTR, and
GLIC on PLA2 activity. To identify the structural domain
of PLA2 involved in interaction with these protein targets,
we studied the inhibitory effect of a specific rattelsnake
PLA2 inhibitor (an oxidiazolone derivative PMS 1062), and
determined the kinetic parameters of inhibition (Ki, Kmapp).
Kinetic characterization of the enzymatic activity of PLA2receptor/channel complexes opens interesting perspectives
to investigate the physiological relevance of these interactions. Co-crystallization studies will also be performed to
determine the binding interface between the protein partners and detect conformational changes after complex formation.
Acknowledgments:
The study was supported by research fellowship within project “Enhancing Educational Potential of Nicolaus Copernicus University in the Disciplines of Mathematical and Natural Sciences (project no. POKL.04.01.0100-081/10.).
References:
Faure G, Saul F (2012) Toxicon 60: 531–538.
P3.26
P3.27
The link between A118G polymorphism
in the μ-opioid receptor (MOR)
gene and predisposition to the
occurrence of addictions
Autogenous activation-repression
role of C protein in Csp231I
restriction-modification system
Konrad Patyra, Katarzyna Regin, Ewa Fiedorowicz,
Michał Matysiewicz, Anna Cieślińska, Elżbieta Kostyra
Anna Petrusewicz, Kinga Ruszkiewicz,
Monika Rezulak, Iwona Mruk
Department of Microbiology, University of Gdansk, Gdańsk, Poland
University of Warmia and Mazury, Department of Biochemistry, Faculty
of Biology and Biotechnology, Olsztyn, Poland
e-mail: Anna Petrusewicz <[email protected]>
e-mail: Konrad Patyra <[email protected]>
Many bacteria produce restriction-modification (R-M)
systems, in part to protect against invading DNA such
as phage genomes. Most type II R-M systems include independently-active restriction endonuclease (REase) and
protective DNA methyltransferase (MTase) proteins. After
R-M genes enter a new cell, MTase activity must appear
before REase or the chromosome will be cleaved. These
R-M systems thus represent temporally-controlled genes,
where the genes have a long-term relationship with their
new hosts, and where the consequences of misregulation
can be lethal.
Some R-M systems achieve delayed REase expression by
co-transcribing the REase gene with the gene for an autogenous transcription regulator (the controlling or “C”
protein). R-M systems associated with C proteins fall into
several groups based on their sequences and target DNA
sequence specificities. The archetypes of three currentlyrecognized groups are the best studied for C.PvuII and
C.EcoRV (names after first example discovered). The
third group — C.EcoO109I comprising two members:
C.EcoO109I and C.Csp231I is unexplored. The purpose
of our research is to investigate C.Csp231I protein and
its R-M system located on chromosome of Citrobacter
sp.RFL231I. Though, the crystal structure of C protein has
been solved, there is no information on biological activity
of C protein in vivo.
C proteins bind to conserved operator sequences called “Cboxes” located upstream of their genes overlapping DNA
promoter region. We tested in vivo effect of C.Csp231I protein on DNA fragments carrying C-boxes fused to reporter
gene when C gene is delivered in trans. The C gene was
cloned under arabinose promoter to obtain a gradient of
C expression after arabinose induction. We observed the
transcription activation in very low C concentration and
subsequent repression in higher C level. The effect was
not present when C protein was mutated to prevent C-box
binding. Same mutation introduced into R-M system background did not abolish the restriction activity suggesting
different mode of regulation of gene expression. In addition, the entire R-M system seems to be toxic to E. coli cells
to such extent the R-M system gene transfer is possible only
if the host cells are premethylated. The host cells show the
distinct filamentation as upon SOS response. Interestingly,
the filament formation is not correlated with the restriction
level, which is unexpected and not clear.
The opioid receptors belong to G-protein coupled family. There are four types of opioid receptors: MOR, NOR,
DOR, KOR. These receptors are mainly located in the
nervous system. Furthermore, they can also be found in
other body tissues such as smooth muscle. They send signals associated with pain perception, motility of the organism or mood control. The opioid receptors also play a role
in the organism’s addiction to opiates, cocaine or alcohol,
especially the μ-opioid receptor (MOR). A high number of
single nucleotide polymorphisms (SNP) has been detected
in the μ-opioid receptor (MOR) gene. One of the most extensively studied SNP is a substitution adenine to guanine
at 118 position which results in the substitution of asparagine to aspartate at 40 position. These mutation occur with
the allele frequency of 10–32% in different ethnics groups.
Some of A118G polymorphism studies suggest that there
is a contribution of the G allele at the increased risk of addiction to heroin and alcohol. Moreover, the occurrence of
G allele may decrease receptor binding ability in smokers.
The aim of the study was to determine the relationship
between A118G polymorphism occurrence in the μ-opioid
receptor (MOR) gene and predisposition to addiction to
different substances (alcohol, energy drink, chocolate, coffee, cigarette) among students. Furthermore, the percentage of genotypes AA, AG and GG in the study population
was estimated.
The questionnaire was distributed among tested students.
Based on the survey, the frequency of drug use was estimated. In addition, the DNA was isolated from students’
blood. The A118G polymorphism of MOR gene was determined using the PCR-RFLP method. The percentage
of examined genotypes was estimated, the obtained results
were correlated with the questionnaire answers.
On the basis of the results, the percentage of genotype AA
was 82%, AG — 14% and GG — 4% in tested population.
Due to the low share of one of the genotypes, there was
no association between the occurrence of the genotype
and predispositions to addiction, but one can notice some
relations. The obtained results indicate on the necessity to
continue working on the μ-opioid receptor (MOR) gene
and its susceptibility to addictions.
52
P3.28
P3.29
Preeclampsia-associated changes in signal
transduction in umbilical cord arteries
Measurement of oxidant-antioxidant
balance with the use of total antioxidant
capacity and total oxidant status
Lech Romanowicz, Zofia Galewska, Tomasz
Gogiel, Krzysztof Sobolewski
Department of Medical Biochemistry, Medical University of Białystok,
Białystok, Poland
e-mail: Lech Romanowicz <[email protected]>
Preeclampsia, the most common pregnancy-associated
pathological syndrome, is accompanied by significant remodelling of the extracellular matrix and alteration in lipid composition of the umbilical cord artery. We evaluate
phosphatidylinositol composition, TGF-β, MAP-kinase
expression, MT-MMP and COX-1, COX-2 activity in
umbilical cord artery and their alteration in preeclampsia.
Solid phase extraction, thin layer chromatography and
high-performance liquid chromatography were employed
for phosphatidylinositol characterization. Western immunoblot and ELISA technique were used for others. The
umbilical cord artery wall is abundant in phosphatidylinositols. Preeclampsia is associated with significant increases
in MT1-MMP and active form of ERK 1/2 content and
proportional activity of COX-2. An increase in MT1-MMP
content may intensify TGF-β conversion to the active form
in preeclamptic umbilical cord arterys. Higher amount of
active form of MAP-kinase may indicate faster signal transduction in comparison to control material. This may cause
a deregulation of cell metabolism, resulting in accumulation of some protein in arterial wall in preeclampsia.
Ewa Romuk1, Bronisława Skrzep-Poloczek1,
Celina Wojciechowska2, Wojciech Jacheć2,
Alina Ostałowska1, Ewa Birkner1
1Silesian Medical University, Department of Biochemistry, 2Silesian
Medical University, Department of Cardiology, Zabrze, Poland
e-mail: Ewa Romuk <[email protected]>
Background: Oxidative stress is induced by a wide range
of environmental factors including UV stress, pathogen invasion and oxygen shortage. Oxygen deprivation stress is
distinguished by three physiologically different states: transient hypoxia, anoxia and reoxygenation. Generation of
reactive oxygen species (ROS) is characteristic for hypoxia
and especially for reoxygenation. The main cellular components susceptible to damage by free radicals are lipids
(peroxidation of unsaturated fatty acids in membranes),
proteins (denaturation), carbohydrates and nucleic acids.
The formation of ROS is prevented by an antioxidant system: low molecular mass antioxidants (ascorbic acid, glutathione, tocopherols), enzymes regenerating the reduced
forms of antioxidants, and ROS-interacting enzymes such
as SOD, peroxidases and catalases. Antioxidants act as a
cooperative network, employing a series of redox reactions.
The measure of total antioxidant capacity (TAC) considers the cumulative action of all the antioxidants present
in plasma and body fluids. The capacity of known and
unknown antioxidants and their synergistic interaction is
therefore assessed, thus giving an insight into the delicate
balance in vivo between oxidants and antioxidants. Measuring plasma TAC may help in the evaluation of physiological, environmental, and nutritional factors of the redox
status in humans. Determining plasma TAC may help to
identify conditions affecting oxidative status in vivo (e.g.,
exposure to reactive oxygen species and antioxidant supplementation).
Total oxidant status (TOS) expresses the total level of all
peroxides produced in the body under the influence of
ROS. It appears to be a more sensitive parameter and much
more accurate than simply marking only the products of
lipid peroxidation.
The aim of this study was estimation and comparison of
TAC and TOS between group of different patients and
searchig the index which is the exponent of the reaction
occurring for maintaining the oxidation-antioxidant balance.
Materials and methods: We have analysed results of
TAC and TOS in 100 patients with osteoarthritis and 190
patients with heart failure and in the control group of 20
healthly volunteers. Total antioxidant capacity (TAC) and
total oxidant status (TOS) was measured with the colorimetric method by Ozcan Erel.
Results: TAC in the control group was 1.25+0.22. TAC in
osteoarthritis group was 0.86+0.14 (p<0.0001, comparing
to the control) and in the heart failure group was 1.25+0.25.
TOS in the control group was 2.24+1.72. TOS in osteoarthritis group was 28.53+0.14 (p0.0001, comparing to
the control) and in the heart failure group was 147.81+0.2
(p<00001, comparing to the control).
Conclusion: Obtained results indicate on the commitment
of oxidation reaction in pathogenesis of osteoartrithis and
heart failure. There was statistically significant increase in
TOS in studied group of patients comparing to the control
group. There was statistically significant decrease of TAC
activity in osteoarthritis patients comparing to the control
and there was no differences between TAC in the heart
failure patients and control. It is seems that antioxidant capacity system is inefficient and isn’t sufficient to protect
organism against ROS.
References:
Erel O (2004) Clin Biochem 37: 277–285.
Blokhina O et al (2003) Ann Bot 179-94.
Haenold R et al (2005) Age (Dordr) 27: 183–199.
P3.30
Structural investigations of equine
and bovine serum albumin binding
properties of 3,5-diiodosalicylic acid
Bartosz Sekula, Kamil Zielinski, Anna Bujacz
Institute of Technical Biochemistry, Lodz University of Technology, Łódź,
Poland
e-mail: Bartosz Sekula <[email protected]>
Transport of different substances within circulatory system is a very intriguing issue especially in a context of metabolites, waste products, toxins and drugs. All these compounds are transported by the most abundant protein of
vertebrates’ plasma — serum albumin. Although all mammalian serum albumins have quite high sequence similarity,
their binding abilities are different.
The heart-shaped and highly helical structure of serum albumin possesses a number of binding pockets. Each of
the three homologous domains has evolved specific binding properties. Although serum albumin domains exhibit
extensive structural homology, the distribution of their
binding sites is not the same.
Salicylic acid and its derivatives are used as components of
pharmaceutical products, dyes, flavors, and preservatives.
The compound, 3,5-diiodosalicylic acid (DIS), is used as
an intermediate for veterinary anthelmintic agents (closantel
and rafoxanide). Due to the presence of iodine atoms, DIS
is also used as a source of heavy atom phases in crystallographic investigations of the new crystal structures of
proteins and, at the same time, is a good ligand for analyzing protein binding properties.
Here we characterize the binding abilities of equine and
bovine serum albumins based on their crystal structures in
complexes with 3,5-diiodosalicylic acid at resolution 2.10 Å
and 2.65 Å, respectively. The comparison of these structures with an analogous complex of human serum albumin
(HSA) reveals numerous differences in the mode of binding of DIS.
Three DIS binding locations are common for this ligand
in BSA and ESA and one site differs. In the case of HSA
there are reports indicating two binding sites of DIS without fatty acids and only one DIS site in the presence of
fatty acids. HSA is unable to bind the 3,5-diiodosalicylic
acid in the locations corresponding to DIS3 and DIS4 in
BSA and ESA, although other aromatic ligands, such as:
iophenoxic acid, indomethacin, iodipamide are capable to
bind in these pockets.
Acknowledgements:
The project is supported by grant No. NN405 363939 from the Polish
Ministry of Science and Higher Education.
References:
1. Bujacz A (2012) Acta Crystallogr D 68: 1278–1289.
2. Sekula B et al (2013) – manuscript under review.
54
P3.31
P3.32
scFv anti-integrin αvβ3 — auristatin E
conjugates in anticancer therapy
Selection of scFv targeting
FGFR1 and FGFR2
Anna M. Serwotka, Jacek Otlewski
Aleksandra Sokołowska-Wędzina,
Aleksandra Borek, Jacek Otlewski
Department of Protein Engineering, Faculty of Biotechnology,
University of Wroclaw, Wrocław, Poland
e-mail: Anna Maria Serwotka <[email protected]>
Despite decades of research, cancer remains a leading
cause of human deaths all over the world. Currently, there
are many recognized cancer markers that can be used both
in diagnostics and targeted therapy. Integrin αvβ3, receptor for vitronectin, belongs to promising cancer markers,
mainly in glioblastoma and melanoma.
Monoclonal antibodies are the most common recombinant
proteins that are applied to cure cancer via recognizing
the cell surface markers of cancer markers. However, full
format antibodies are large proteins (~150 kDa) that are
rather difficult and expensive to produce. Instead, smaller
antibody fragments, like scFvs (single-chain variable fragments), can be used. scFvs are the smallest antibody fragments that are capable of antigen recognition. An additional advantage of scFvs is the possibility of production in
bacterial systems. The ease of production of scFv proteins
is also reflected in the much lower cost of production.
In this study, scFv anti-integrin αvβ3 was designed based on
sequence of humanised lapin monoclonal antibody antiintegrin αvβ3. scFv protein was produced in mammalian
CHO-S cell line and purified on Ni-NTA agarose. scFv was
properly folded, as revealed by circular dichroism spectra.
The specificity of scFv – integrin αvβ3 interaction was confirmed by surface plasmon resonance, flow cytometry analysis and fluorescence microscopy. Experiments with U-87
MG glioblastoma cell line overexpressing integrin αvβ3
showed that scFv specifically recognizes integrin αvβ3. No
interaction was observed in negative control experiments
on SK-BR-3 adenocarcinoma cell line.scFv anti-integrin
αvβ3 — MMAE (auristatin E) conjugate was synthesized
and its cytotoxicity will be evaluated in the forthcoming
experiments.
Acknowledgements:
The research was supported by Wroclaw Research Centre EIT+ under the
project „Biotechnologies and advanced medical technologies” – BioMed
(POIG.01.01.02-02-003/08) financed from the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2).
University of Wroclaw, Department of Protein Engineering, Wrocław,
Poland
e-mail: Aleksandra Sokołowska-Wędzina <[email protected]>
Fibroblast Growth Factor Receptors 1 and 2 (FGFR1 and
FGFR2) overexpression has been found in different types
of human cancer, including breast, bladder, prostate, endometrial, lung and hematologic cancers. Thus, FGFRs are
promising candidates for potential cancer targeting molecule. To selectively target FGFR 1 and 2 overexpressed
in cancer we used the strategy based on antibody targeting. Antibodies are the most specific and common protein
binders, which provide high affinity and specificity to the
interaction with the target. To generate FGFR 1 and 2 specific antibodies we used phage display technology. Due to
the large size of immunoglobulin, we chose Tomlinson library based on antibody fragments: scFv scaffold (scFv —
single chain Fragment variable).
Using Tomlinson I and J library, we selected a panel of antibody fragments specific for FGFR1 and FGFR2. After 3 or
4 rounds of selection we performed monoclonal ELISA of
selected clones. At this stage we obtained 41 positive clones
interacting with FGFR1, and 25 positive clones interacting
with FGFR2. We further verified their potential interaction
with receptors using another technique: SPR (Surface Plasmon Resonance) screening. We confirmed positive interaction
for 20 (anti-FGFR1) and 15 (anti-FGFR2) ELISA-positive
clones with their target receptors. Sequence analysis of
SPR-positive clones has identified 18 different sequences
of scFv selected against FGFR1, and 5 unique sequences
selected against FGFR2. We found specific amino acid motifs within CDR3 loop of heavy chain, which is a region
particularly important in antigen binding. Finally we chose
5 representative scFv variants for FGFR1, and 5 scFv variants for FGFR2, which were then expressed in HB2151
cells and purified using Protein A Sepharose. SPR measurements were performed with different concentrations
of scFv monomeric fraction. Two strongest FGFR1 —
specific binders, and one FGFR2 — specific binder were
chose for further affinity maturation. 48th Congress of the Polish Biochemical Society
P3.33
P3.34
CMP-sialic acid/UDP-N-acetylglucosamine
chimeric protein partially improves
glycosylation in mammalian cells
deficient in UDP-galactose and
CMP-sialic acid transporters
ApoLp-III-derived polypeptides from
Galleria mellonella hemolymph
Paulina Sosicka, Dorota Maszczak-Seneczko, Mariusz Olczak
Laboratory of Biochemistry, Faculty of Biotechnology, University of
Wrocław, Wrocław, Poland
e-mail: Paulina Sosicka <[email protected]>
Glycosylation is one of the most important posttranslational modification which occurs in the endoplasmic reticulum
and Golgi apparatus. Plenty of enzymes are involved in this
process whilst the most important are transferases, which
deliver sugars to the glycan structure and nucleotide sugar
transporters (NSTs), which provide substrates for them.
Studies on the function and substrate specificity of NSTs
includes structural modifications leading to the development of chimeric proteins, composed of portions derived
from different transporters. In literature two such NSTs
have already been described. Both CMP-sialic acid (CST;
SCL35A1)/UDP-galactose (UGT; SLC35A2) and UDPgalactose/UDP-N-acetylglucosamine (NGT; SLC35A3)
chimeric proteins were constructed to determine part of
the transporter crucial for its substrate specificity. In our
present study we investigated a novel chimeric transporter
which is composed of the N-terminal portion of human
CST and the C-terminal part of human NGT. CST/NGT
was overexpressed in three cell lines, which are well known
from their glycosylation defects — MDCK-RCAr, CHOLec8 (cell lines defficient in UGT) and CHO-Lec2 (cell line
defficient in CST). We udes UGT deficient cells because in
our previous study we have found that NGT overexpression in these cell lines partially restore galactosylation of Nglycans. Using immunohistochemical staining we observed
that CST/NGT chimeric protein was localized in the Golgi
apparatus of stably transfected cells. Further experiments
in CHO-Lec2 cells performed by application of highly
specific lectins have shown that this transporter partially
rescues sialylation defect. Moreover, there is slight improvment on galactosylation and sialylation in UGT deficient
cells. This data suggest that CST/NGT chimeric protein
might be able to transport UDP-galactose and CMP-sialic
acid in some extent but they still need to be confirmed via
structutal glycans analysis and investigation of UDP-galactose and CMP-sialic acid uptake into the Golgi apparatus.
Aneta Sowa-Jasiłek1, Paweł Mak2, Sylwia Stączek1,
Agnieszka Zdybicka-Barabas1, Małgorzata Cytryńska1
1Institute of Biology and Biochemistry, Department of Immunobiology,
Maria Curie-Skłodowska University, Lublin, Poland; 2Faculty of
Biochemistry, Biophysics and Biotechnology, Jagiellonian University,
Kraków, Poland
e-mail: Aneta Sowa-Jasiłek <[email protected]>
Apolipophorin III (apoLp-III) is an abundant hemolymph
protein involved in immune response and lipid transport in
insects. This protein is able to associate with bacterial and
fungal cells through binding components of their cell walls,
e.g. lipopolysacharides, lipoteichoic acids and β-1,3-glucan.
Moreover, upon bacterial challenge, apoLp-III interacts
with lipids and joins to lipophorin particles forming low
density lipophorins, which are absorbed by insect blood
cells — hemocytes. ApoLp-III is involved in pathogen
recognition, in detoxification of microbial cell wall components and can be considered as a signaling molecule informing the insect immune system about infection. It was
demonstrated that apoLp-III can exist in hemolymph in
several forms differing in isoelectric point.
Our previous research revealed that apoLp-III (18 kDa)
occurs in two main forms in the hemolymph of naive
Galleria mellonella larvae, but after immune challenge additional three or four forms (depending on the immunogen
used) differing in pI and molecular mass were detected. As
apoLp-III is synthesized in a fat body and was also detected
in the hemocytes, we analyzed if apoLp-III-derived polypeptides are present in these tissues of naive individuals
and larvae immunized by Gram-negative bacteria (Escherichia coli), Gram-positive bacteria (Micrococcus luteus), yeasts
(Canida albicans) and filamentous fungi (Fusarium oxysporum).
Immunoblotting performed with anti-apoLp-III antibodies
after IEF/SDS-PAGE revealed one form of apoLp-III in
the fat body of naive as well as immune-challenged insects,
whereas in the hemocytes usually two forms of the protein
differing in isoelecteric point were detected.
G. mellonella apoLp-III-derived polypeptides present in
hemolymph of larvae challenged by different pathogens
were separated using RP-HPLC chromatography. Two
fractions were received: the first fraction contained 14 kDa
and 7 kDa polypeptides, while the second fraction contained differing in pI apoLp-III isoforms of ca. 18 kDa.
The percentage ratio of the first to the second fraction in
the hemolymph varied depending on the microorganism
used to immune-challenge of the larvae. Our findings may
indicate that apoLp-III is synthesized in the fat body as one
of the basic form of the protein, but in the hemocytes and
mainly in the hemolymph after infection various forms of
apoLp-III appear in response to attack of different pathogens. The detected apoLp-III-derived polypeptides and
isoforms probably are generated from apoLp-III by limited proteolysis, which occurs in the hemolymph and the
hemocytes after infection. It is possible that in response
to particular class of pathogen characteristic pattern of
apoLp-III-derived polypeptides are produced.
56
P3.35
P3.36
Novel primosomal protein B from
Clostridium pasteurianum
Characterization of the novel cysteine
petidase Tpr from the pathogenic
bacterium Porhyromonas gingivalis W83
and its interaction with the cystatins
— human host protease inhibitors
Marta Śpibida, Marta Marszałkowska, Marcin Olszewski
Gdańsk University of Technology, Department of Microbiology, Gdańsk,
Poland
e-mail: Marta Śpibida <[email protected]>
Dominika Staniec1,2, Magnus Abrahamson2, Jan Potempa1,3
PriB is a primosomal protein that catalyzes DNA replication in Procaryota. The replication pathway starts with
PriA protein — the initiator protein that binds to a DNA
replication fork, unwinds double-stranded DNA and role
of PriB is to stabilize PriA on the DNA. However there are
many biochemical differences in replication mechanism in
bacteria and only some of them use PriB proteins. A few
of PriB proteins were published and only three structures
of them were resolved (Escherichia coli, Klebsiella pneumoniae
and Neisseria gonorrhoeae). All up-to-date known PriB proteins have one OB domain per monomer and they are homodimers in solution.
Recently, we have published the crystal structure of PriB
protein from Thermoanaerobacter tengcongensis that represents
new class of PriB with two oligonucleotide/oligosaccharide-binding domain (OB) per monomer that means it exists as monomer in solution.
The aim of this study is identification and characterization of the primosomal protein B (PriB) from bacterium
Clostridium pasteurianum (CpaPriB). It is the largest known
bacterial PriB protein consisting 234 amino acid residues
with a calculated molecular mass of 30 kDa. Surprisingly,
it is functional as monomer containing two single-stranded
DNA binding domain (OB-fold) and it is the completely
new kind structure of SSB protein like PriB protein from
thermophilic bacterium Thermoanaerobacter tengcongensis.
Therefore, our studies suggest that we discovered new
classes of PriB from mesophilic bacteria.
1Department
of Microbiology, Faculty of Biochemistry, Biophysics, and
Biotechnology, Jagiellonian University, Kraków, Poland; 2Department of
Laboratory Medicine, Division of Clinical Chemistry and Pharmacology,
Lund University, Sweden; 3University of Louisville Dental School, Center
for Oral Health and Systemic Diseases, Louisville, USA
e-mail: Dominika Staniec <[email protected]>
The relation between the pathogenic potential of P. gingivalis, an etiologic agent of periodontitis, and other serious
illnesses like cardiovascular and lung diseases or rheumatoid arthritis is confirmed. Peptidases produced by P. gingivalis are recognized as essential virulence factors. Accordingly, bioinformatics analysis of the P. gingivalis genome
revealed the presence of several genes encoding putative peptidases. One of these genes (PG1055) encodes a
protein (‘Tpr’, thiol protease) with sequence similarity to
cysteine peptidases of the calpain family. We have already
created a large-scale expression and purification system,
which allows to obtain recombinant protein Tpr. The
peptidase showed low latency and underwent autocatalytic
processing. Examination of proteolytic activity of Tpr released that enzyme possess Ca2+-dependent activity against
gelatin, Z-Phe-Arg-NHMec, and Suc-Leu-Leu-Val-TyrNHMec, keeping with calcium-dependent autoproteolysis. These results confirmed that enzyme recognizes and
digests amino acid sequences typical for collagen and might
play role in host destructive events in periodontities. To investigate the possibility that human-derived natural cysteine
peptidase inhibitors can inhibit bacterial calpain-like peptidase, we determined the effect of cystatin A and C on the
Tpr activity, and found that cystatins are potent inhibitors.
As the peptidases can be obvious target for drug design in
novel therapy strategies in order to dealing with periodontal
disease, this results could aid in development of synthetic
inhibitors, potential therapeutic agents. Moreover, to our
knowledge Tpr is first bacterial cysteine protease which is
inhibited by cystatins. This project would enable also further progress into investigation of more complex systems,
e.g. peptidases — inhibitors models.
P3.37
P3.38
Application of fluorescent Bocillin for
testing penicillin binding properties of
recombinant PBP 3, PBP 4 and PBP 5
proteins from Neisseria meningitidis RNA chaperone activity of proteins
essential for the life cycle of HIV2: implications for the design of
the retroviral inhibitors.
Karol Stawski1, Anna Skoczyńska2, Anna Goc1
Katarzyna Pachulska-Wieczorek, Agnieszka K. Stefaniak,
Katarzyna J. Purzycka, Ryszard W. Adamiak
1Department of Genetics, Nicolaus Copernicus University, Toruń,
Poland; 2Department of Epidemiology and Clinical Microbiology.
National Medicines Institute, Warsaw, Poland
e-mail: Karol Stawski <[email protected]>
RNA Structure and Function Laboratory, Institute of Bioorganic
Chemistry, Polish Academy of Sciences, Poznań, Poland
e-mail: Agnieszka Katarzyna Stefaniak <[email protected]>
Neisseria meningitidis is a Gram-negative human pathogen
and a leading cause of bacterial meningitis and septicemia worldwide. A reduced susceptibility or resistance to
penicillin is connected in some bacteria with alterations in
penicillin binding proteins (PBPs), a group of enzymes responsible for the final periplasmic steps in the synthesis
and maturation of peptidoglycan. The varied PBPs occur
in different numbers per bacterial cell and have variable affinities for penicillin. For example a gram-positive bacteria
Bacillus subtilis have 16 PBPs while gram-negative Neisseria
meningitidis only 5. The differences between sequences of
low molecular mass PBP 3, PBP 4 and PBP 5 of N. meningitides may affect their enzymatic properties such as DDcarboxypeptidase or endopeptidase activity. We have amplified selected genes encoding PBPs of N. meningitidis using
oligonucleotide primers and PCR products were cloned
into pEt28a plasmid. The recombinant soluble PBP 3, PBP
4 and PBP 5 proteins do not include signal peptides, additionally PBP 3 and PBP5 are lacking extreme 22 and 105
amino acids of carboxyl terminus, respectively. Plasmids pEt28a encoding recombinant PBPs were transformed
into E. coli Bl21(DE3)pLysS and their expression was induced with 1 mM IPTG for 4 hrs. Next, cells were pelleted
and PBPs containing polyhistidine-tag were extracted using
Ni2+ affinity chromatography columns.The interaction of
beta-lactam with a recombinant PBPs was assessed by incubating the proteins for 0,5, 1, 2, 4 and 8 min with Bocillin, a
fluorescent penicillin. The fluorescence emission from Bocillin in SDS gel of PBP 3, PBP 4 and PBP 5 have revealed
their high affinity to beta-lactam antibiotics. However the
rate of Bocillin acylation by recombinant PBPs was very
fast and saturation of the enzymes occurred in the first 30
seconds.
Acknowledgements:
This work was supported by Nicolaus Copernicus University in Toruń
(research grant no. 1371-B).
While preventive vaccine against AIDS is still not available,
conventional therapies including highly active retroviral
therapy (HAART), are based primarily on the HIV reverse
transcriptase and protease inhibitors. This approach is associated with a number of side effects due to its high toxicity. Identification of the molecular mechanisms of HIV
retroviruses, with particular emphasis on the structure and
properties of macromolecules involved, is an indispensable prerequisite for the design of effective therapies against
HIV/AIDS.
Nucleocapsid proteins (NC) are crucial for the HIV
genomic RNA dimerization, packaging and reverse transcription, making these proteins new targets for the antiretroviral therapy. However, such an approach has not yet
been exploited because of the lack of sufficient knowledge
concerning both, specific and non-specific interactions of
NC with viral RNA. Released from Gag, during viron maturation, nucleocapsid proteins (NC) are small basic proteins that possess two copies of highly conserved CCHC
zinc fingers. The NC functions in HIV replication are correlated with their ability to act as nucleic acids chaperones
(NAC). In contrast to NCp7 protein (HIV-1) little is known
about the NAC activity of NCp8 (HIV-2).
Our research is focused on identifying interactions of
HIV-2 RNA with regulatory proteins Tat-2, NCp8 and
Gag polyprotein, that are crucial for the HIV replication.
We examined the ability of NCp8 to chaperone annealing
of DNA with complementary strand as well as DNA and
RNA strand exchange in duplex nucleic acids in vitro. Furthermore, we investigated whether NCp8 is capable of enhancing ribozyme-directed cleavage of an RNA substrate
in vitro. We found that NCp8 possesses nucleic acids chaperone activity similar to but somewhat different than NCp7
protein.
58
P3.39
P3.40
The influence of resveratrol and
its derivatives on the structure
and function of glyceraldehyde
3-phosphate dehydrogenase under
oxidative stress conditions
Structural and thermodynamic
characteristics of avidinbiotinylruthenocene complex
Joanna Strumiłło, Aleksandra Rodacka, Mieczysław Puchała
1Institute of Technical Biochemistry, Lodz University of Technology,
Łódź, Poland; 2Department of Organic Chemistry, University of Lodz,
Łódź, Poland
Institute of Biophysics, Department of Molecular Biophysics, Division of
Radiobiology, University of Łódź, Łódź, Poland e-mail: Joanna Strumiłło <[email protected]>
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
is glycolytic enzyme ubiquitous both in eukaryotic and
prokaryotic proteomes. It is responsible for the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. GAPDH is also one of the classical examples of
multifunctional proteins called "moonlighting" proteins. It
plays a key role in such processes as transcription, apoptosis and vesicle shuttling. Furthermore, oxidatively modified
GAPDH is found to be involved in the protein aggregation
processes and pathogenesis of neurodegenerative diseases.
Resveratrol and its derivatives are naturally occurring polyphenolic compounds with anti-oxidative properties. It is
believed that they have anticancer, cardioprotective properties and prevents from neurodegeneration.
The objective of the presented research was to examine
the influence of natural polyphenols on the structure and
function of glyceraldehyde-3-phosphate dehydrogenase in
the presence or absence of hydrogen peroxide. The compounds used in this study were: resveratrol and its analog
(trans-3,3,5,5-tetrahydroksy-4-metoksystilbene), yuccaol A
and yuccaol C.
We investigated the influence of the above mentioned compounds on the activity, secondary structure and content of
thiol groups in the presence or absence of hydrogen peroxide. Furthermore, we checked the rate of 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid) fluorescence after binding
to protein exposed to polyphenols both in the presence
and absence of hydrogen peroxide. There was no significant influence of the used compounds on the activity and
thiols content in the absence of hydrogen peroxide. On the
other hand, during oxidative stress induced by hydrogen
peroxide and in the presence of used polyfenols we observed inactivation of the enzyme, changes in the content
of free thiol groups and secondary structure of GAPDH.
However, no significant increase of 1,8-ANS fluorescence
was observed. The most significant changes in the secondary structure were observed in the presence of hydrogen
peroxide and resveratrol analog and with both yuccaols.
The presented data clearly indicate that resveratrol, its
analog and yuccaols A and C have pro-oxidative properties
toward glyceraldehyde-3-phosphate dehydrogenase.
Paweł Strzelczyk1, Anna Bujacz1, Damian Plażuk2,
Janusz Zakrzewski2, Grzegorz Bujacz1
e-mail: Paweł Strzelczyk <[email protected]>
Avidin is a glycosylated protein from eggs of amphibians,
reptiles and birds. Homotetramer of avidin binds four molecules of biotin with the highest known in Nature affinity
(Kd ≈ 10-13- 10-15 M). This interaction is several orders of
magnitude higher than in the typical antigen-antibody complexes [1]. The biotin-avidin system, based on the extremely high affinity is widely used in bioanalysis, drug delivery
and nanotechnology.
This study is focused on the interaction of avidin from hen
egg white with biotinylruthenocene (BR) based on crystallographic research and isothermal titration calorimetry. We
present crystal structure of avidin-BR complex (PDB ID:
4I60) and thermodynamic parameters of interaction between these two molecules.
Avidin is organized in eight-stranded antiparallel β-barrel
with right-handed twist. Crystal of the avidin-BR complex
belongs to a tetragonal system in the space group P42212.
The tetrameric assembly is located at the crossing of the
three two-fold axes and is created by crystal symmetry.
Each monomer is stabilized in the tetramer by interactions of extended loops and the external surfaces of the
β-barrels. The crystal structure of the avidin-BR complex
shows that most of the interaction of biotin moiety is
maintained in comparison to avidin–biotin complex and
the ruthenocene moiety is capable of interacting with protein via hydrogen bonds and π-stacking contacts. The larger
ligand practically does not influence the avidin conformation with the exception of a small movement of the loop
L3-4. The biotin conjugation with ruthenocene reduced ligand affinity, which was expected. The replacement of the
strong hydrogen bonds created between the carboxyl group
from biotin with Ser73 and Ser75 by weaker contacts with a
cyclopentadienyl ring from the biotinylruthenocene moiety
could account for the lower affinity of that ligand to avidin.
Smaller affinity of the biotinylruthenocene to avidin may
be beneficial when such complex will be used as a drug
delivery system [2]. References:
1. Rosano C et al (1999) Biomolecular Engineering 16: 5–12.
2. Strzelczyk P et al (2013) Chemico-Biological Interactions 204: 6–12.
Acknowledgements:
This research was supported by grant 2012/05/B/NZ1/00701 from the
National Science Centre.
P3.41
P3.42
Effect of ferulic acid on fungal 26S
proteasome activities in vitro
Proline-rich polypeptide complex
(prp) and it’s nonapeptide fragment
(np) protect neuronal cells from
toxic effect of amyloid beta 1-42
Anita Swatek, Anna Golecka, Magdalena Staszczak
Department of Biochemistry, Institute of Biology and Biochemistry, Maria Curie-Skłodowska University, Lublin, Poland
e-mail: Anita Swatek <[email protected]>
The 26S proteasome, a large multicatalytic protease complex is involved in highly selective ATP-dependent and
ubiquitin-mediated degradation of intracellular proteins in
eukaryotes. Its presence was found in animals, plants and
yeast [1, 2]. In the kingdom of fungi proteasomes were
also reported in ligninolytic Basidiomycetes [3, 4]. The proteasome-mediated proteolysis plays a key role in the regulation of critical cellular processes such as transcriptional
control, cell cycle progression, oncogenesis, and stress response. Previous studies from our laboratory demonstrated
involvement of ubiquitin/proteasome pathway in the regulation of ligninolytic activities in the wood-decaying fungi.
The 26S proteasome is assembled from a core particle (20S
proteasome) and one or two regulatory complexes 19S. The
core particle of the 26S proteasome houses proteolytically
active sites, identified based on their specificity towards
short synthetic peptides as: “chymotrypsin-like” (CHTL), “trypsin-like” (T-L), and “caspase-like” (C-L). In the
present work we studied effect of ferulic acid (4-hydroxy5-methoxycinnamic acid), known as an inducer of some
ligninolytic enzymes, on peptidase activities of the 26S
proteasomes isolated from mycelia of the white-rot fungus
Trametes versicolor. Proteasomes were separated from other
low molecular proteases using Biomax PBVK polyethersulfone ultrafiltration membranes (NMWL 500 kDa) [3].
Peptidase activities were detected by monitoring cleavage
of fluorogenic substrates: Suc-LLVY-AMC, Z-GGR-AMC
and Z-LLE-βNA for CHT-L, T-L, and C-L activity, respectively. Moreover, the ability of ferulic acid (in concentrations up to 2 mM) to affect degradation of a model protein
substrate (β-casein) by the isolated 26S proteasomes was
also evaluated using spectrofluorimetric and electrophoretic analyses. The absolute rate of protein degradation was
spectrofluorimetrically quantified by the modified fluorescamine assay [5] for release of free α-amino groups generated upon the cleavage of peptide bonds in β-casein.
Acknowledgments:
This work was partly supported by the Polish Scientific Project BS/
UMCS.
Agnieszka Zabłocka1, Anna Urbaniak1,
Marianna Kuropatwa2, Maria Janusz1
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences, 1Laboratory of Immunochemistry,
2Laboratory of Signaling Proteins, Wrocław, Poland
e-mail: Anna Urbaniak <[email protected]>
Introduction: Deposits of amyloid β protein are pathological hallmark of AD. They are toxic for neural cells both
in vivo and in vitro. Aggregates and fibrilar forms of amyloid
β are closely associated with inflammation. It was shown in
several reports that uncontrolled production of nitric oxide
could be involved in the amyloid β42 toxicity.
Aim: The ability of proline-rich polypeptide complex
(PRP) isolated from ovine colostrum and its nonapeptide
fragment (NP) to protect neuronal cell line PC12 from toxic effects of aggregated amyloid β42 was studied.
Methods: Rat pheochromocytoma PC12 cell line was
treated with PRP/NP and aggregated amyloid β42. PRP/
NP was applied to the cells simultaneously, 24 hours before
and 24 hours after amyloid β42 treatment. Cell viability was
checked by MTT assay. The level of neuronal NOS was determined by Western Blotting. The intracellular level of nitric oxide was measured by Griess method after reduction
of nitrates to nitrites using NADPH – dependent nitrate
reductase. The level of cGMP was detected by ELISA.
Results: A colostral proline-rich polypeptide complex PRP
and its nonapeptide fragment (NP) can modulate the toxic
effect of amyloid β42 on neuronal PC 12 cell line. It was
observed that PRP and NP significantly lowered the toxicity of amyloid β42. The regulatory effect of PRP/NP on
NO production induced by amyloid β42 was also shown.
The neuronal response to amyloid β42 was cGMP-dependent only for PRP when used at low doses.
Conclusion: The results obtained indicate that both PRP
and NP can modulate the amyloid β42 cytotoxicity.
Acknowledgments:
This research work was supported by the Ministry of Sciences and High
Education, Poland (project number N N302217838).
References:
1. Ciechanover A (2005) Nat Rev Mol Cel Biol 6: 79–86.
2. Vierstra R (2003) Trends Plant Sci 8: 135–142.
3. Staszczak M, Jarosz-Wilkołazka A (2005) Biochimie 87: 755–762.
4. Staszczk M (2008) 45: 328–337.
5. Kisselev AF, Goldberg AL (2005) Methods Enzymol 398: 364–378.
60
P3.43
P3.44
Construction and selection of phage
display libraries based on variable
lymphocyte receptor sequences
VEGF in intraluminal thrombus
of abdominal aorta
Magdalena Wezner-Ptasińska, Jacek Otlewski
University of Wrocław, Department of Protein Engineering, Wrocław,
Poland
e-mail: Magdalena Wezner-Ptasińska <[email protected]>
Macromolecules specifically recognizing certain antigens
are invaluable tools in research and clinical applications.
The most common natural binders are antibody molecules
providing high affinity and specificity towards the target.
Recently, the alternative binders, usually small and stable
proteins, have challenged the immunoglobulin applications.
Usually, binding molecules can be selected from combinatorial libraries based on display technologies. Phage display
is a powerful methodology that allows the selection of a
particular phenotype (e.g., binding to a defined antigen)
from repertoires of proteins displayed on phage surface.
Over the years it has been established as a robust and reliable technology for specific binders selection.
In the project the alternative scaffold based phage display
libraries were tested as a source of proteins specifically recognizing biomarkers for cancer therapies. Two different phage display libraries were constructed on the previously
designed dVLR (variable lymphocyte receptor) template
based on receptor sequences that mediate adaptive immunity in sea lamprey. dVLR contains six LRR (leucine-rich
repeat) modules forming horseshoe shape structure whose
concave surface is responsible for target binding. Crystal
structure analysis of VLR-ligand complexes indicated the
positions responsible for interaction with antigens. For
these positions different schemes of randomization have
been chosen. Two different phage display libraries has been
designed: P16 — 16 randomized positions and Sm11S —
11 randomized positions, resulting in functional library size
of 2×1010 and 1×108 amino acid sequences respectively.
In both libraries SRP protein translocation pathway has
been applied. In next step phage display libraries were used
for isolation of binders. Cancer biomarkers S100A7 and
HER2 were chosen as targets for panning. A wide panel of
selection protocols was tested, both on plastic surface and
in solution. Preliminary surface plasmon resonance experiments confirmed interaction between selected variants and
desired antigen.
Acknowledgements:
„The research was supported by Wroclaw Research Centre EIT+ under
the project „Biotechnologies and advanced medical technologies” – BioMed (POIG.01.01.02-02-003/08) financed from the European Regional
Development Fund (Operational Programme Innovative Economy,
1.1.2)”.
Małgorzata Wolańska1, Emilia Bańkowska-Guszczyn1,
Radosław Kowalewski2, Krzysztof Sobolewski1
1Department of Medical Biochemistry and 2Department of Vascular
Surgery & Transplantation, Medical University of Bialystok, Białystok,
Poland
e-mail: Małgorzata Wolańska <[email protected]>
The pathogenesis of aorta aneurism is characterized by a
breakdown of extracellular matrix due to an excessive proteolytic activity. First of all elastin is degraded and replaced
by collagen. The remodelling of arterial wall is controlled
by the action of some peptide growth factors, especially by
VEGFs. In most cases the aorta aneurisms are accompanied by intraluminal thrombi (ILT). The functions of cytokines, especially those of VEGFs, in the pathobiology of
arterial wall are relatively well known. On the other hand
the peptide growth factors in the intraluminal thrombi
were not studied till now. For this reason it was decided to
search for the expression of VEGFs, their receptors and
mitogen-induced kinases (MAP)-kinases) in intraluminal
thrombi taken from patients submitted to surgical treatment of abdominal aneurism aorta.
Studies were performed on fragments of aneurism walls
taken from 24 patients (20 men and 4 women) of the age
62–80 (average 67.9±7.2) operated at the Department of
Vascular Surgery and Transplantation. The diameter of
the aneurism was 72±14 mm. The parietal thrombi were
separated from aneurism wall and divided into luminal and
abluminal layers.The control material (aorta wall) was the
fragments of abdominal aorta taken from 12 organ donors
(10 men and 2 women) of the age 47±5.
The most important finding of our study was demonstration the high expression of VEGFs in both layers of intraluminal thrombus. No doubt the blood-borne cells are engaged in their biosynthesis. Also the receptors of VEGFs
were detected. Furthermore, the activity of phosphorylated MAP-kinases: ERK1 and ERK-2 was found in intraluminal thrombus. In some cases the expression of VEGFs
and their receptors in the thrombus was even higher that in
aorta wall and in aneurism wall. It allows to conclude that
some blood cells may synthesizes VEGFs which stimulate
these cells to produce extracellular enzymes which degrade
the fibrin. Furthermore, the VEGF may penetrate from the
thrombus to aneurism wall and to enhance the neovascularisation process. Futhermore it may stimulate the remodelling of extracellular matrix. It may result in enhanced degradation of elastin and its replacement by collagen.
P3.45
P3.46
Studies on specificity of DNA
methyltransferase M.BstZ1II
and its deletion mutant
Major urinary protein levels in
cystathionine beta synthase deficient mice
Ewa Wons-Karczyńska, Iwona Mruk, Tadeusz Kaczorowski
Department of Microbiology, University of Gdańsk, Gdańsk, Poland
e-mail: Ewa Wons-Karczyńska <[email protected]>
DNA methylation is a major form of epigenetic modification. The process is provided by DNA methyltransferases
(MTases), which catalyze a transfer of methyl group from
the donor S-adenosyl-L-methionine to the adenine or cytosine residue within recognized nucleotide sequence. MTases
fall into two groups depending on methylated atom — endocyclic MTases (C-Mtases) modify carbon C5 of cytosine,
while exocyclic MTases (N-MTases) methylate nitrogen
from amino group of adenine (N6) or cytosine (N4).
MTases contain two domains — the larger with a set of
nine/ten well defined amino acids motifs creating the
AdoMet binding pocket and the active site, and the smaller
— responsible for recognizing the specific DNA sequence
(TRD — target recognition domain). While larger domains
of all MTases share a common core structure referred as
AdoMet-dependent MTase fold, smaller domains of different proteins are dissimilar in amino acids sequence, size
and structure.
We have been focusing on the group of five MTases, which
despite origin in distinct bacteria species recognize the
same nucleotide sequence 5’-AAGCTT-3’ and modify the
first adenine residue at N6. These enzymes are: M.HindIII,
M.EcoVIII, M.Csp231I, M.LlaCI and M.BstZ1II. Comparison of their amino acid sequences revealed 68-aa region located before TRD in M.BstZ11II, which was neither
present in the rest of studied proteins, nor was similar to
any other sequences in the GenBank. Therefore the mutant protein devoid the region was constructed and named
M.BstZ1IIΔ. Since the plasmid DNA carrying shorter variant of M.BstZ1II was fully protected against digestion by
restriction endonuclease of the same specificity, the mutant protein was active. To determine the possible role of
the region, the specificity towards different substrate DNA
of two purified variants (mutant and WT) was assessed.
Both forms showed relaxed specificity when reaction mixture was supplemented with 40% DMSO and were able
to modify sequences with single substitutions in regard to
the canonical AAGCTT. M.BstZ1IIΔ exhibited slightly
stronger preference towards the 2nd position of the specific
sequence than the WT. Moreover, it appeared that the 68aa region may be responsible for interacting with the DNA
backbone, as M.BstZ1IIΔ modified with lower efficiency
all substrates lacking internucleotide linkage within the canonical sequence than the WT as well as in contrast to the
WT was unable to methylate canonical sequence located
directly at the end of the oligonucleotide.
Jacek Wróblewski1, Joanna Perła-Kaján2,
Hieronim Jakubowski1,2,3
1Poznań University of Life Sciences, Department of Biochemistry and
Biotechnology, Poznań, Poland; 2UMDNJ-New Jersey Medical School,
International Center for Public Health, Department of Microbiology and
Molecular Genetics, Newark, NJ 07101, USA; 3Institute of Bioorganic
Chemistry, Poznań, Poland
e-mail: Jacek Wróblewski <[email protected]>
Background: Major Urinary Proteins (MUP) are excreted
in mouse urine and play an important role in sexual signaling by binding volatile pheromones and prolonging their
persistence in scent marks. MUPs expression decreases in
senescent males. Female mice tend to avoid senesced, older
mates, which are unable to retain the ability to produce
costly sexual signals. Factors affecting MUPs expression
are largely unknown. Here, we examined how hyperhomocysteinemia due to cystathionine β-synthase (Cbs) deficiency impacts the expression of MUPs in the mouse.
Methods: We used the C57BL/6J Tg-I278T Cbs-/- mouse
model (Jakubowski H et al., 2009, FASEB J 23 :1721–177).
MUPs concentrations and urinary creatinine in Cbs-/- mice
(n=7) and Cbs+/+ littermates (n=5) were determined using Coomassie and picric acid, respectively. Patterns of
MUP expression were analyzed on SDS/PAGE gels. Urinary homocysteine (Hcy), Hcy-thiolactone, and N-Hcyprotein levels were measured using HPLC-based assays
(Jakubowski H, 2008, Anal Biochem 380: 257–261) on 1260
Infinity UPLC instrument (Agilent). Site-specific N-homocysteinylation of MUPs lysine residues was analyzed using
LC-MS/MS.
Results: Urinary Hcy, Hcy-thiolactone, and N-Hcy-protein
levels were extremely elevated in Cbs-/- mice, compared to
Cbs+/+ animals. Cbs-/- male mice had significantly lower urinary MUP (protein/creatinine ratio was 0.38±0.13 in Cbs-/mice and 0.93±0.08 in Cbs+/+ animals), seen also on SDSPAGE gels. Patterns of MUPs separated on SDS-PAGE
gels differed between Cbs-/- and Cbs+/+ mice. LC-MS/MS
analysis of tryptic digests of MUPs from male and female
Cbs-/- mice identified a MUP11 peptide, 95AG(N-Hcy-K)
YSVTYDGFNTF108, containing Hcy N-linked to lysine
residue K97.
Conclusions: Hyperhomocysteinemia increases N-homocysteinylation and decreases expression of MUPs in
mice. These findings identify MUPs as a new target for
N-homocysteinylation in vivo and suggest that hyperhomocysteinemic males are impaired in their ability to produce
scent marks, and thus may be less preferred by their females mates.
62
P3.47
P3.48
Lithium chloride protects neurons
from toxicity of cytosolic PrP
Crystallographic studies of serum
albumins in complexes with ligands
Tomasz Zajkowski, Hanna Nieznanska, Krzysztof Nieznanski
Kamil Zielinski, Bartosz Sekula, Anna Bujacz
Department of Biochemistry, Nencki Institute of Experimental Biology,
Warsaw, Poland Institute of Technical Biochemistry, Faculty of Biotechnology and Food
Sciences, Lodz University of Technology, Łódź, Poland
e-mail: Tomasz Zajkowski <[email protected]>
e-mail: Kamil Zieliński <[email protected]>
Prion protein (PrP) is mostly extracellular glycoprotein anchored in plasma membrane, constitutively expressed in
the nervous system. In misfolded state this protein is prone
to form aggregates, which are found in diseases called
transmissible spongiform encephalopathies (TSE). In
some of these diseases PrP abnormally localizes in cytosol (cytoPrP) interacting with proteins, which are not its
physiological partners. In previous studies we have demonstrated that PrP is causing tubulin oligomerization, inhibits microtubule formation and disassembles microtubular
cytoskeleton of epithelial cells. We have also showed that
microtubule associated proteins (MAPs) that regulate microtubule stability can prevent above-mentioned effect of
PrP. According to our study at least two proteins of this
group: Tau and MAP2 where able to prevent deleterious
effect of PrP but not in phosphorylated state. Interestingly,
hyperphosphorylated Tau has been frequently detected in
TSE. Extending the application of the above one should
be able to modulate effect of cytoPrP on microtubular
cytoskeleton by influencing level of phosphorylation of
MAPs. In our study we used synthetic peptide encompassing first 30 amino acids of PrP (PrP1-30) as a model of
cellular prion protein. The peptide consists of N-terminal
signal sequence responsible for penetration of the molecule through cell membrane and major tubulin-binding site
(23–30). To reduce level of phosphorylation of MAPs we
chose inhibitors of GSK-3 a kinase known to be responsible for modification of these proteins. Specific inhibitor of the kinase — CT98014 and less selective LiCl were
employed. By means of confocal microscopy we demonstrated disassembly of microtubular cytoskeleton and loss
of neurites of primary neurons exposed to PrP1-30. In
contrast, the cells treated with PrP1-30 in the presence of
either LiCl or CT98014 did not differ from control cells. In
cytotoxicity tests (MTT, LDH) we confirmed protection of
neurons from toxic effect of PrP1-30 by both inhibitors of
GSK-3. Our observations may help in understanding the
molecular mechanism of neurotoxicity of cytoPrP. Since
LiCl is well-known pharmaceutical used in the treatment of
several mental disorders we believe that our study may be
useful in design of therapy of TSE.
Serum albumins are highly soluble proteins naturally present in plasma. Due to their unique properties and high
ability to bind various ligands, mammalian serum albumins
are used in many laboratory procedures. They are known
as versatile transport proteins that carry physiological ligands such as: fatty acids, hormones, bilirubin and others
[1].
Most of the serum albumin binding sites have affinity to
more than one group of ligands, therefore determination
of the protein-ligand interactions are essential for understanding the complex character of this protein transporter.
Albumin is a heart-shaped molecule composed of three
helical domains consisting of two subdomains each, showing a different binding specificity.
The topology of serum albumin binding pockets has been
well characterized based on the human serum albumin
(HSA) crystal structures and it has been explored in other
species [2].
The inspiration for the presented research was the observation that drug affinity could be dependant on the presence
of other small molecules that bound to albumin.
We present the crystal structures of bovine, equine and
leporine serum albumin complexes with an iodine derivative of salicylic acid and naproxen. The first ligand is used
as an intermediate for veterinary anthelmintic agents, a
medication capable of causing the evacuation of parasitic
intestinal worms, while the second is a non-steroidal antiinflammatory drug, commonly used for the reduction of
pain, fever and inflammations.
Comparison of the investigated complexes will allow to determine the differences in ligand binding properties, which
result from different sequence and spatial specificity of
individual albumins. The structural investigations of these
complexes provide the information about their surface
shape and potential that can be utilized as specific epitopes
for antibodies.
Acknowledgements:
The project is supported by grant No. NN405 363939 from the Polish
Ministry of Science and Higher Education.
References:
1. Petitpas I et al (2001) J Mol Biol 314: 955–960.
2. Bujacz A (2012) Acta Cryst Biol Crystallogr D68: 1278–1289.
P3.49
Yeast prion protein Rbs1
Marta Żurańska
Institute of Biochemistry and Biophysics PAS, Department of Genetics,
Warsaw, Poland
e-mail: Marta Żurańska <[email protected]>
Prions (protein infectious particles) are unusual proteins
that exist stably in many functionally distinct conformations
at least one of which is self-replicating. Prions are responsible for several severe mammalian illnesses like Creutzfeldt–
Jakob disease or bovine spongiform encephalopathy. On
the contrary in yeasts they do not cause diseases, but act as
non-Mendelian inheritance elements that have important
role in such processes as fidelity of translation termination
or transcription regulation. Yeast prions are thought to be
mechanism for generating fast phenotypic diversity that
helps to survive in changing environment. Rbs1 has been
identified among the candidates selected in a bioinformatic
proteome-wide survey for prionogenic proteins in Saccharomyces cerevisiae (Alberti et al., 2009, Cell 137: 146–158).
N-terminal part of Rbs1 protein of ordered structure contains R3H domain which potentially binds single stranded
nucleic acids. In contrast, C-terminal part of Rbs1 forms a
molten globule.
Prion features of Rbs1 are supported by our current studies. We found that yeast Rbs1 protein shuttles between the
cytoplasm and the nucleus. When localised in cytoplasm,
Rbs1 forms foci. The amount of foci is increased in the
older the cells. Inactivation of R3H domain by mutagenesis decreases efficiency of foci formation. Rbs1 is not essential protein and its exact function is still unknown but
our work indicates its influence on biogenesis of RNA
polymerase III.

Session 3. Proteins — Structure and Function Lectures L3.1 L3.2

Transkrypt

Podobne dokumenty

Survey - S. O. S. Europe

Surviving WZT-1 Armoured Recovery Vechicle

Computational modeling of the bacterial 70S ribosome and its

3.1 Session 3. Application of natural products in human and ani

Structural Relationships Among the Ribosomal Stalk Proteins from